Nucleic acid constructs and methods of using the same

ABSTRACT

Described herein are block copolymers, and methods of making and utilizing such copolymers. The described block copolymers are disruptive of a cellular membrane, including an extracellular membrane, an intracellular membrane, a vesicle, an organelle, an endosome, a liposome, or a red blood cell. Preferably, in certain instances, the block copolymer disrupts the membrane and enters the intracellular environment. In specific examples, the block copolymer is endosomolytic and capable of delivering an oligonucleotide (e.g., an mRNA) to a cell. Compositions comprising a block copolymer and an oligonucleotide (e.g., an mRNA) are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/827,793, filed Nov. 30, 2017, which is a continuation of U.S.application Ser. No. 14/908,351, filed Jan. 28, 2016, which is theNational Stage of International Application No. PCT/US14/48839, filedJul. 30, 2014, now issued as U.S. Pat. No. 9,867,885, which claims thebenefit of U.S. Application No. 61/868,122, filed Aug. 21, 2013, andU.S. Application No. 61/860,136, filed Jul. 30, 2013, each of which isincorporated in its entirety by reference.

FIELD

This invention relates to the fields of organic chemistry, polymerchemistry, biochemistry, molecular biology and medicine. Moreparticularly, this invention relates to copolymers, conjugates ofcopolymers with oligonucleotides, and complexes of copolymers witholigonucleotides to be used for delivery of oligonucleotides into cells.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted inASCII format via EFS-Web to the United States Patent and TrademarkOffice. Said ASCII Copy, created on Aug. 16, 2019, is named“4170_0030004_SL_ST25” and is 113,271 bytes in size. Due to theelectronic filing of the Sequence Listing, the electronically submittedSequence Listing serves as both the paper copy required by 37 C.F.R. §1.821(c) and the CRF required by 37 C.F.R. § 1.821(e). The informationcontained in the Sequence Listing is hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION

Various diseases today require a treatment which involves administrationof peptide-, protein-, and nucleic acid-based drugs, particularly thetransfection of nucleic acids into cells or tissues. The fulltherapeutic potential of peptide-, protein-, and nucleic acid-baseddrugs is currently compromised by their limited ability to cross theplasma membrane of mammalian cells, resulting in poor therapeuticefficacy.

RNA molecules have the capacity to act as potent modulators of geneexpression in vitro and in vivo and therefore have great potential asnucleic acid based drugs. These molecules can function through a numberof mechanisms utilizing either specific interactions with cellularproteins or base pairing interactions with other RNA molecules. RNAinterference is a process of gene silencing that plays an important rolein development and maintenance of the genome. The RNAi pathway iscomplex. It is initiated by the enzyme dicer which cleaves doublestranded RNA (dsRNA) into fragments. An RNA-induced silencing complex(RISC) is then formed by base pairing between complementary mRNA and theguide strand of each new fragment. The passenger strand of each fragmentis degraded. This formation of the RISC complex leads to translationalsilencing or degradation of the complementary mRNA by the endonucleaseargonaute. Argonaute is the catalytic component of the complex. Theshort fragments are known as small interfering RNA (siRNA) and microRNA(miRNA) for example. Modulation of gene expression via RNA effectormolecules, such as siRNA, has great therapeutic potential as themodulatory complexes formed, be they RNA-protein complexes or RNA-RNAcomplexes, are often highly specific. However, in order for such RNAeffector molecules to modulate gene expression they must be present inthe cell's cytoplasm to enter into the RISC Complex.

The delivery of exogenous oligonucleotides such as RNA molecules andother membrane impermeable compounds into living cells is highlyrestricted by the complex membrane systems of the cell. Typically,molecules used in antisense and gene therapies are large, negativelycharged and hydrophilic molecules. These characteristics preclude theirdirect diffusion across the cell membrane to the cytoplasm. For thisreason, the major barrier to the therapeutic use of oligonucleotides formodulation of gene expression is the delivery of the oligonucleotide tothe cytoplasm. Transfection agents used in the art today typicallycomprise peptides, polymers, and lipids of a cationic nature as well asnano- and microparticles. These transfection agents typically have beenused successfully only in in vitro reactions as the cationic nature ofthese systems, while facilitating both cell binding and binding of theoligonucleotide, renders them ineffective or toxic in vivo. Furthermore,the cationic charge of these systems causes interaction with serumcomponents, which causes destabilization of theoligonucleotide-transfection reagent interaction and poorbioavailability and targeting. When transfecting nucleic acids in vivofurther requirements have to be fulfilled. For example, the complexshould not interact with parts of the complement system of the host.Additionally, the complex should protect the nucleic acid from earlyextracellular degradation by ubiquitously occurring nucleases.Furthermore, the carrier should not be recognized by the adaptive immunesystem (immunogenicity) and should not stimulate an acute immuneresponse.

Although high transfection efficiencies are possible in vitro, achievingsimilar extents of transfection without toxicity is difficult in vivo.In general, exogenous unmodified nucleic acid molecules, particularlyviral nucleic acids, introduced into the cell induce an innate immuneresponse which results in cytokine and interferon (IFN) production andultimately cell death. It is of great interest for therapeutics,diagnostics, reagents and for biological assays to be able to deliver anucleic acid, e.g., a ribonucleic acid (RNA), into a cell, such as tocause intracellular translation of the nucleic acid and production ofthe encoded protein instead of generating an innate immune response.This delivery issue is currently the major prohibitive factor for theapplication of nucleic acid-based drugs, particularly RNA basedtherapeutics, in vivo. Thus, there remains a need for an effectivedelivery system for efficiently delivering nucleic acid-based drugs,particularly RNA based therapeutics, to cells and tissues. The presentinvention provides compositions and methods for the delivery and releaseof an oligonucleotide to a cell.

BRIEF SUMMARY

The present disclosure provides block copolymers for the effectivedelivery of an oligonucleotide to a cell. The present disclosure alsoprovides for methods of using the block copolymers, methods of treatmentusing the block copolymers, processes for preparing the block copolymersand pharmaceutical compositions including the block copolymers.

In one example, the disclosure provides a block co-polymer of theformula IT1-L1-[A]_(x)-[B]_(y)-Z  Iwhere

-   T1 is absent or a first targeting moiety;-   L1 is absent or a linking moiety;    A is a first block that is a polymer formed from monomers comprising    formula A2 or a random copolymer formed from monomers comprising    formulae A1, A2 and A3; A1 and A2; A2, A4 and A5; A2 and A5; or A4    and A5;

where R¹ is H or C₁-C₆ alkyl, R² is O, S, NH, N(C₁-C₆ alkyl), or(OCH₂CH₂)₁₋₁₂₀, and Q is selected from the group consisting of (i)S-S-pyridyl, (ii) S-S-G, (iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where Vis an amide, ester, imine, oxime, thioester, product of a [3+2]cycloaddition, product of a [4+1] cycloaddition, carbonate, carbamate,urea, acetal, ketal, or hydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀,C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), (viii) S-S-L2-G wherein L2 is

-   where n=1-35 and    designates a point of attachment of L2 to G,-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation;

where n is 1-120, R³ is H or C₁-C₆ alkyl, R⁴ is S, O, NH or N(C₁-C₆alkyl), R⁵ is O or S and R⁶ is H, C₁-C₆ alkyl, C₁-C₆ alkyl-NH₂, C₁-C₆alkyl-NH(C₁-C₆ alkyl), C₁-C₆ alkyl-N(C₁-C₆ alkyl)₂;

where R⁷ and R¹⁰ are independently H or C₁-C₆ alkyl, R⁸ is S, O, NH orN(C₁-C₆ alkyl), and R⁹ is O or S and R¹¹ is an amine protecting group;

where n is 1-230, R¹⁷ is H or C₁-C₆ alkyl, R¹⁸ is O, S, NH or N(C₁-C₆alkyl), R¹⁹ is O or S, and R²⁰ is OH, NH, H, T2, or C₁-C₆ alkyl, whereT2 is a second targeting moiety;

where R²¹ is H or C₁-C₆ alkyl, R²² is O, NH or N(C₁-C₆ alkyl), R²³ is H,aryl, arylhalide, alkyl, alkyl alcohol;

B is a second block that is a random copolymer formed from monomerscomprising formulae B1, B2, B3 and B4 or B1, B2 and B3

where R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are independently H or C₁-C₆alkyl, R¹⁸ is O, S, NH, N(C₁-C₆ alkyl), or (OCH₂CH₂)₁₋₁₂₀, and Q isselected from the group consisting of (i) S-S-pyridyl, (ii) S-S-G, (iii)(OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide, ester, imine,oxime, thioester, product of a [3+2] cycloaddition, product of a [4+1]cycloaddition, carbonate, carbamate, urea, acetal, ketal, or hydrazone,and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, orthioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and

-   (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation;-   x is 2-20 kDa;-   y is 2-20 kDa;-   Z is H, SH, C(CH₃)₂CN,

where R²⁴ is S—(C₁-C₁₂ alkyl), aryl, arylhalide, O—(C₁-C₁₂ alkyl),NR²⁵R²⁶ where R²⁵ and R²⁶ are independently H, alkyl, aryl, orheteroaryl;

-   the ratio of x to y is from 2:1 to 1:4; and-   designates a point of attachment.

In some embodiments of a copolymer of Formula I above, the monomer offormula A2 is

where n is 1-20, R³ is H or C₁-C₆ alkyl, R⁴ is S, O, NH or N(C₁-C₆alkyl), R⁵ is O or S and R⁶ is H, C₁-C₆ alkyl, C₁-C₆ alkyl-NH₂, C₁-C₆alkyl-NH(C₁-C₆ alkyl), C₁-C₆ alkyl-N(C₁-C₆ alkyl)₂.

In another example, the disclosure provides a method for theintracellular delivery of an oligonucleotide comprising: a) contacting ablock copolymer of Formula I as described above, where G is present andis an oligonucleotide, with a cell where the copolymer is introducedinto an endosomal membrane within the cell through endocytosis; and b)destabilizing the endosomal membrane, whereby the copolymer or theoligonucleotide is delivered to the cytosol of the cell.

In another example, the disclosure provides a method of treatinghepatocellular carcinoma, cholangiocarcinoma, hepatitis,hypercholesterolemia, liver fibrosis, pulmonary fibrosis orhaemochromatosis comprising administering to a mammal in need thereof atherapeutically effective amount of a block copolymer of Formula I asdescribed above, wherein Q is S-S-oligonucleotide,

and

designates a point of attachment.

In another example, the disclosure provides a pharmaceutical compositioncomprising a block copolymer of Formula I as described above and apharmaceutically acceptable diluent or carrier, wherein Q isS-S-oligonucleotide,

and

designates a point of attachment.

In yet another example, the disclosure provides a pharmaceuticalcomposition comprising a (a) block copolymer of Formula I wherein G ispresent and is cationic peptide, (b) an mRNA molecule, and (c) apharmaceutically acceptable diluent or carrier.

In yet another example, the disclosure provides a process forpreparation of the polymer of Formula I as described above including thesteps of:

a) contacting a compound of Structure Va, Vb, Vc, or Vd,

where R²⁷═C₁-C₁₂ alkyl,

where R²⁸═C₁-C₁₂ alkyl,

where R²⁵ and R²⁶ are independently H, alkyl, aryl, or heteroaryl,

where T1 is absent or a first targeting moiety and L1 is absent or alinking moiety; with one or more monomers selected from monomers of theformulae A1, A2 and A3,

where R¹ is H or C₁-C₆ alkyl, R² is O, S, NH, N(C₁-C₆ alkyl), or[O(CH₂CH₂)]₁₋₁₂₀, Q is —SR²⁰ or S-S-pyridyl, and R²⁰ is athiol-protecting group;

where n is 1-120, R³ is H or C₁-C₆ alkyl, R⁴ is S, O, NH or N(C₁-C₆alkyl), R⁵ is O or S and R⁶ is H, C₁-C₆ alkyl, C₁-C₆ alkyl-NH₂, C₁-C₆alkyl-NH(C₁-C₆ alkyl), C₁-C₆ alkyl-N(C₁-C₆ alkyl)₂;

where R⁷ and R¹⁰ are independently H or C₁-C₆ alkyl, R⁸ is S, O, NH orN(C₁-C₆ alkyl), and R⁹ is O or S and R¹¹ is an amine protecting group;in the presence of a free radical;

-   b) contacting the product of step a) with monomers of formulae B1,    B2 and B3,

where R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H or C₁-C₆ alkyl; inthe presence of a free radical; and c) deprotecting the product of stepb) and contacting it with an oligonucleotide, cationic peptide,polyamine, or polycation comprising a thiol-reactive or amine-reactivegroup; or contacting the product of step b) with an oligonucleotide,cationic peptide, polyamine, or polycation comprising a thiol group. Insome embodiments where the product of step b) is contacted with acationic peptide, polyamine, or polycation comprising a thiol-reactiveor amine-reactive group, or with a cationic peptide, polyamine, orpolycation comprising a thiol group, the process further includescontacting the product of step c) with a polynucleotide (e.g., an mRNA)to form a complex comprising the block copolymer of Formula I and thepolynucleotide. In particular variations of a method as above, R²⁵and/or R²⁶ is a heteroaryl having the structure:

In some embodiments of a method as above, for the monomer of formula A2,n is 1-20.

In yet another example, the disclosure provides a process forpreparation of the polymer of Formula I as described above including thesteps of:

-   a) contacting a compound of Structure Va, Vb, Vc, or Vd,

where R²⁷═C₁-C₁₂ alkyl,

where R²⁸═C₁-C₁₂ alkyl,

where R²⁵ and R²⁶ are independently H, alkyl, aryl, or heteroaryl,

where T1 is absent or a first targeting moiety and L1 is absent or alinking moiety; with one or more monomers selected from monomers of theformulae A2, A4 and A5,

where n is 1-120, R³ is H or C₁-C₆ alkyl, R⁴ is S, O, NH or N(C₁-C₆alkyl), R⁵ is O or S and R⁶ is H or C₁-C₆ alkyl;

where R¹⁷ is H or C₁-C₆ alkyl, R¹⁸ is O, S, NH or N(C₁-C₆ alkyl), R¹⁹ isO or N, R²⁰ is H, T2, or C₁-C₆ alkyl, where T2 is a second targetingmoiety;

where R²¹ is H or C₁-C₆ alkyl, R²² is O, NH or N(C₁-C₆ alkyl), R²³ is H,aryl, arylhalide, alkyl, alkyl alcohol;

-   b) contacting the product of step a) with monomers of formulae B1,    B2, B3, and B4,

where R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are independently H or C₁-C₆alkyl, R¹⁸ is O, S, NH or N(C₁-C₆ alkyl), and Q is —SR²⁰ or S-S-pyridyl,and R²⁰ is a thiol-protecting group; and c) deprotecting the product ofstep b) and contacting it with an oligonucleotide, cationic peptide,polyamine, or polycation comprising a thiol-reactive or amine-reactivegroup; or contacting the product of step b) with an oligonucleotide,cationic peptide, polyamine, or polycation comprising a thiol group. Insome embodiments where the product of step b) is contacted with acationic peptide, polyamine, or polycation comprising a thiol-reactiveor amine-reactive group, or with a cationic peptide, polyamine, orpolycation comprising a thiol group, the process further includescontacting the product of step c) with a polynucleotide (e.g., an mRNA)to form a complex comprising the block copolymer of Formula I and thepolynucleotide. In particular variations of a method as above, R²⁵and/or R²⁶ is a heteroaryl having the structure

In some embodiments of a method as above, for the monomer of formula A2,n is 1-20.

In yet another example, the disclosure provides a compound of theformula

These and other examples will be apparent from a reading of thefollowing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The disclosure may be more completely understood in consideration of thefollowing detailed description of various embodiments of the disclosurein connection with the accompanying Figures, in which:

FIG. 1 is a graph demonstrating human β-catenin mRNA knockdown relativeto human MET mRNA after β-catenin siRNA (si033)/polymer dosing.

FIG. 2 is a graph demonstrating human MET mRNA knockdown relative tohuman β-catenin mRNA after MET siRNA (si034)/polymer dosing.

FIG. 3 is a graph demonstrating knockdown of human β-catenin mRNA andhuman MET mRNA upon the administration of a combination formulation ofβ-catenin and MET siRNAs with polymer.

FIG. 4 is a graph demonstrating the knockdown of β-catenin protein byβ-catenin siRNA following β-catenin siRNA (si033)/polymer treatment.

FIG. 5 is a graph demonstrating the knockdown of MET protein followingβ-catenin siRNA (si033)/polymer treatment.

FIG. 6 is a graph demonstrating the knockdown of MET protein by METsiRNA following MET siRNA (si034)/polymer treatment.

FIG. 7 is a graph demonstrating the knockdown of β-catenin protein byMET siRNA following MET siRNA (si034)/polymer treatment.

FIG. 8 is a graph demonstrating ALT levels through 96 hr post siRNAdosing of formulations of si033 (β-catenin siRNA) with polymer, si034(MET siRNA) with polymer or a combination of si033 and si034 withpolymer.

FIG. 9 is a graph demonstrating ALT levels 6 and 10 days post siRNAdosing of formulations of si033 (β-catenin siRNA) with polymer, si034(MET siRNA) with polymer or a combination of si033 and si034 withpolymer.

FIGS. 10A and 10B depict exemplary structures of linking moiety L1.

FIGS. 11A-11D depict exemplary structures of targeting moiety T1 linkedto linking moiety L1 (T1-L1-together).

FIGS. 12A-12C depict exemplary block copolymers of Formula I.

FIGS. 13A and 13B schematically depict the synthesis of an exemplaryblock copolymer in two polymerization steps: a first block (conjugationblock) polymerization (FIG. 13A) and a second block (endosome releaseblock) polymerization (FIG. 13B).

DETAILED DESCRIPTION

The present invention is directed to copolymers, compositions, andmethods useful for delivering oligonucleotides or other cell-impermeablemolecules to mammalian cells. The following detailed description is notto be taken in a limiting sense. Unless defined otherwise, all technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art pertinent to the methodsand compositions described. The definitions provided herein are tofacilitate understanding of certain terms used frequently herein and arenot meant to limit the scope of the present disclosure.

As used herein the term “alkyl” as used herein refers to a monovalentstraight or branched hydrocarbon of from 1 to 12 carbon atoms. Examplesof alkyl groups include, but are not limited to methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, andn-hexyl.

As used herein, the term “block copolymer” refers to two or morehomopolymer or copolymer subunits linked by covalent bonds. Blockcopolymers with two or three distinct blocks are called diblockcopolymers and triblock copolymers, respectively. A schematicgeneralization of a diblock copolymer is represented by the formula[F_(f)G_(g)H_(h) . . . ]_(q)-[J_(j)K_(k)L_(l) . . . ]_(r), wherein eachletter stands for a constitutional unit derived from polymerization of acorresponding monomer and wherein each subscript to a constitutionalunit represents the mole fraction of that unit in the particular block,the three dots indicate that there may be more (there may also be fewer)constitutional units in each block and q and r indicate the molecularweight of each block in the diblock copolymer. As suggested by theschematic, in some instances, the number and the nature of eachconstitutional unit is separately controlled for each block. Theschematic is not meant and should not be construed to infer anyrelationship whatsoever between the number of constitutional units andthe number of different types of constitutional units in each of theblocks. Nor is the schematic meant to describe any particular number orarrangement of the constitutional units within a particular block. Ineach block the constitutional units may be disposed in a purely random,an alternating random, a regular alternating, a regular block or arandom block configuration unless expressly stated to be otherwise. Apurely random configuration, for example, may have the non-limitingform: f-f-g-h-f-g-g-h-g-h-h-h . . . . An exemplary alternating randomconfiguration may have the non-limiting form: f-g-f-h-g-f-g-h-g-f-h . .. , and an exemplary regular alternating configuration may have thenon-limiting form: f-g-h-f-g-h-f-g-h . . . . An exemplary regular blockconfiguration may have the following non-limiting configuration: . . .f-f-f-g-g-g-h-h-h-f-f . . . , while an exemplary random blockconfiguration may have the non-limiting configuration:f-f-f-h-h-f-f-g-g-g-h-h-h-f-f-h-h-h . . . . In a gradient polymer, thecontent of one or more monomeric units increases or decreases in agradient manner from the α-end of the polymer to the ω-end. In none ofthe preceding generic examples is the particular juxtaposition ofindividual constitutional units or blocks or the number ofconstitutional units in a block or the number of blocks meant nor shouldthey be construed as in any manner bearing on or limiting the actualstructure of block copolymers described herein. As used herein, thebrackets enclosing the constitutional units are not meant and are not tobe construed to mean that the constitutional units themselves formblocks. That is, the constitutional units within the square brackets maycombine in any manner with the other constitutional units within theblock, i.e., purely random, alternating random, regular alternating,regular block or random block configurations. The block copolymersdescribed herein are, optionally, alternate, gradient or random blockcopolymers.

As used herein, the term “molecular weight” for a polymer or polymerblock is the number average molecular weight. It is understood in theart that a population of polymer molecules will have a distribution ofdifferent molecular weights. This distribution of molecular weights canbe described by the term dispersity index or polydispersity index (PI orPDI), which is the weight average molecular weight/number averagemolecular weight.

As used herein, the term heteroaryl is an aromatic heterocyclic ring.The heteroatom in a heteroaryl can be O, N, or S. Examples of heteroarylinclude pyridyl or pyridine, imidazole, and oxazole.

As used herein, the term “antibody” refers to any immunoglobulin proteinthat specifically binds to an antigen, as well as antigen-bindingfragments thereof and engineered variants thereof. Hence, the term“antibody” includes, for example, polyclonal antibodies, monoclonalantibodies, and antigen-binding antibody fragments that contain theparatope of an intact antibody, such as Fab, Fab′, F(ab′)₂ and F(v)fragments. Genetically engineered intact antibodies and fragments, suchas chimeric antibodies, humanized antibodies, single-chain Fv fragments,single-chain antibodies, diabodies, minibodies, linear antibodies,multivalent or multispecific hybrid antibodies, and the like are alsoincluded. Thus, the term “antibody” is used expansively to include anyprotein that comprises an antigen binding site of an antibody and iscapable of binding to its antigen. In some embodiments, an antibody hasaffinity to a cell surface molecule.

The term “genetically engineered antibodies” means antibodies whereinthe amino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques in the generationof antibodies, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with cells andother effector functions. Typically, changes in the variable region willbe made in order to improve the antigen binding characteristics, improvevariable region stability, or reduce the risk of immunogenicity.

An “antigen-binding site of an antibody” is that portion of an antibodythat is sufficient to bind to its antigen. The minimum such region istypically a variable domain or a genetically engineered variant thereof.Single-domain binding sites can be generated from camelid antibodies(see Muyldermans and Lauwereys, J. Mol. Recog. 12:131-140, 1999; Nguyenet al., EMBO J. 19:921-930, 2000) or from V_(H) domains of other speciesto produce single-domain antibodies (“dAbs”; see Ward et al., Nature341:544-546, 1989; U.S. Pat. No. 6,248,516 to Winter et al.). In certainvariations, an antigen-binding site is a polypeptide region having only2 complementarity determining regions (CDRs) of a naturally ornon-naturally (e.g., mutagenized) occurring heavy chain variable domainor light chain variable domain, or combination thereof (see, e.g., Pessiet al., Nature 362:367-369, 1993; Qiu et al., Nature Biotechnol.25:921-929, 2007). More commonly, an antigen-binding site of an antibodycomprises both a heavy chain variable domain and a light chain variabledomain that bind to a common epitope. Examples of molecules comprisingan antigen-binding site of an antibody are known in the art and include,for example, Fv fragments, single-chain Fv fragments (scFv), Fabfragments, diabodies, minibodies, Fab-scFv fusions, bispecific(scFv)₄-IgG, and bispecific (scFv)₂-Fab. (See, e.g., Hu et al., CancerRes. 56:3055-3061, 1996; Atwell et al., Molecular Immunology33:1301-1312, 1996; Carter and Merchant, Curr. Opin. Biotechnol.8:449-454, 1997; Zuo et al., Protein Engineering 13:361-367, 2000; andLu et al., J. Immunol. Methods 267:213-226, 2002.)

As used herein, the terms “single-chain Fv” and “single-chain antibody”refer to antibody fragments that comprise, within a single polypeptidechain, the variable regions from both heavy and light chains, but lackconstant regions. In general, a single-chain antibody further comprisesa polypeptide linker between the V_(H) and V_(L) domains, which enablesit to form the desired structure that allows for antigen binding.Single-chain antibodies are discussed in detail by, for example,Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113(Rosenburg and Moore eds., Springer-Verlag, New York, 1994), pp.269-315. (See also WIPO Publication WO 88/01649; U.S. Pat. Nos.4,946,778 and 5,260,203; Bird et al., Science 242:423-426, 1988.)Single-chain antibodies can also be bi-specific and/or humanized.

As used herein the term “oligonucleotide” refers to a polymer comprising7-20,000 nucleotide monomeric units (i.e., from 7 nucleotide monomericunits to 20,000 nucleotide monomeric units, inclusive). Typicaloligonucleotides in accordance with certain embodiments of the presentinvention include those comprising 7-20,000 nucleotide monomeric units,7-15,000 nucleotide monomeric units, 7-10,000 nucleotide monomericunits, 7-5,000 nucleotide monomeric units and 7-1000 nucleotidemonomeric units. Oligonucleotides include deoxyribonucleic acid (DNA)and ribonucleic acid (RNA), or their derivatives, and combinations ofDNA, RNA. DNA may be in form of cDNA, in vitro polymerized DNA, plasmidDNA, parts of a plasmid DNA, genetic material derived from a virus,linear DNA, vectors (P1, PAC, BAC, YAC, and artificial chromosomes),expression vectors, expression cassettes, chimeric sequences,recombinant DNA, chromosomal DNA, anti-sense DNA, or derivatives ofthese groups. RNA may be in the form of messenger RNA (mRNA), in vitropolymerized RNA, recombinant RNA, transfer RNA (tRNA), small nuclear RNA(snRNA), ribosomal RNA (rRNA), chimeric sequences, dicer substrate andthe precursors thereof, locked nucleic acids, anti-sense RNA,interfering RNA (RNAi), asymmetric interfering RNA (aiRNA), smallinterfering RNA (siRNA), microRNA (miRNA), ribozymes, external guidesequences, small non-messenger RNAs (snmRNA), untranslatedRNA (utRNA),snoRNAs (24-mers, modified snmRNA that act by an anti-sense mechanism),tiny non-coding RNAs (tncRNAs), small hairpin RNA (shRNA), or theirderivatives. In addition, DNA and RNA may be single, double, triple, orquadruple stranded. Double stranded RNA (dsRNA) and siRNA are ofinterest particularly in connection with the phenomenon of RNAinterference. Examples of oligonucleotides as used herein include, butare not limited to, siRNA, an antisense oligonucleotide, a dicersubstrate, a miRNA, an aiRNA or an shRNA. Further examples ofoligonucleotides as used herein include, but are not limited to dsRNAhaving a length of from 17 to 29 nucleotides, or from 19 to 25nucleotides, and being at least 90 percent, or 95 percent or 100 percent(of the nucleotides of a dsRNA) complementary to a coding or anon-coding section of the nucleic acid sequence of a therapeuticallyrelevant protein or antigen. Ninety percent complementary means that a20 nucleotide length of a dsRNA contains not more than 2 nucleotideswithout a corresponding complementarity with the corresponding sectionof the mRNA. Yet further examples of oligonucleotides as used hereininclude, but are not limited to single stranded mRNA which can bemodified or unmodified. Modified mRNA includes those with at least twomodifications and a translatable region. The modifications may belocated on the backbone and/or a nucleoside of the nucleic acidmolecule. The modifications may be located on both a nucleoside and abackbone linkage. Typically, mRNAs in accordance with certaincompositions and methods of the present invention include thosecomprising 300-20,000 nucleotide monomeric units, 300-15,000 nucleotidemonomeric units, 300-10,000 nucleotide monomeric units, 300-5,000nucleotide monomeric units, 300-2000 nucleotide monomeric units,300-1,500 nucleotide monomeric units, and 300-1000 nucleotide monomericunits. In some variations, an mRNA in accordance with compositions andmethods of the present disclosure is at least 500, at least 1,000, atleast 1,200, or at least 1,500 nucleotide monomeric units (e.g., from500 to 20,000 nucleotide monomeric units; from 1,000 to 20,000nucleotide monomeric units; from 1,200 to 20,000 nucleotide monomericunits; from 1,500 to 20,000 nucleotide monomeric units; from 500 to15,000 nucleotide monomeric units; from 1000 to 15,000 nucleotidemonomeric units; from 1,200 to 15,000 nucleotide monomeric units; from1,500 to 15,000 nucleotide monomeric units; from 500 to 10,000nucleotide monomeric units; from 1,000 to 10,000 nucleotide monomericunits; from 1,200 to 10,000 nucleotide monomeric units; from 1,500 to10,000 nucleotide monomeric units; from 500 to 5,000 nucleotidemonomeric units; from 1,000 to 5,000 nucleotide monomeric units; from1,200 to 5,000 nucleotide monomeric units; from 1,500 to 5,000nucleotide monomeric units; from 500 to 2,000 nucleotide monomericunits; from 1,000 to 2,000 nucleotide monomeric units; or from 1,200 to2,000 nucleotide monomeric units).

As used herein the term “cationic peptide” refers to a polymercomprising 2-100 amino acid monomers whose overall charge is positive.

As used herein, the term “polycation” refers to a moiety having positivecharges at a plurality of sites and whose overall charge is positive.Examples of polycations include but are not limited to spermine,spermidine, pentaethylenehexamine, tetraethylenepentamine,1,4-bis(3-aminopropyl)piperazine, linear or branched polyethyleneimine,chitosan, polyvinylamine, poly(vinylpyridine), and amino cyclodextrins.

As used herein, a “targeting moiety” refers to a moiety that is capableof specifically binding to (i) a molecule on the surface of a targetcell or (ii) a molecule that is capable of specifically binding to amolecule on the surface of a target cell, such as a cell within a targettissue of a subject. A molecule (e.g., cell surface molecule) thatspecifically binds to a targeting moiety is also referred to herein as a“binding partner.” In some embodiments of copolymers and relatedcompositions and methods as described herein, a targeting moietyspecifically binds to a molecule on the surface of the target cell.Particularly suitable targeting moieties include antibodies,antibody-like molecules, polypeptides, proteins (e.g., insulin-likegrowth factor II (IGF-II)), peptides (e.g., an integrin-binding peptidesuch as an RGD-containing peptide), and small molecules such as, forexample, sugars (e.g., lactose, galactose, N-acetyl galactoseamine(NAG), mannose, mannose-6-phosphate (M6P)) or vitamins (e.g., folate).In some variations, a targeting moiety is a protein derived from anatural ligand of a cell-surface molecule (e.g., derived from a cytokineor from the extracellular domain of a cell-surface receptor that bindsto a cell surface counter-receptor). Examples of cell surface moleculesthat may be targeted by a targeting moiety of a copolymer providedherein include, but are not limited to, the transferrin receptor type 1and 2, the EGF receptor, HER2/Neu, VEGF receptors, integrins, NGF, CD2,CD3, CD4, CD8, CD19, CD20, CD22, CD33, CD43, CD38, CD56, CD69, theasialoglycoprotein receptor, mannose receptor, and thecation-independent mannose-6-phosphate/IGF-II receptor.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Some proteins are defined herein interms of their amino acid backbone structures.

As used herein the term “peptide” refers to a polypeptide having 2-100amino acid monomers.

A polypeptide that is a targeting moiety (e.g., T1 and/or T2) is alsoreferred to herein as a “targeting polypeptide.” A “targeting peptide”is a targeting polypeptide that has 2-100 amino acid monomers.Typically, targeting polypeptides as used herein target or delivercopolymers to target cells or tissues, or specific cells types andenhance the association of molecules with the target cells. Examples oftargeting polypeptides as used herein include, but are not limited to,signal peptides, cell penetrating peptides such as TAT or KALA forexample, integrin-binding peptides such as RGD-containing peptides, NL4,neurotensin, secretin, LOX-1 binding insulin, EGF, IGF-II, GE7 andtransferrin. In some embodiments, a targeting polypeptide is asingle-chain antibody.

As used herein the term “sugar” refers to saccharides such asmonosaccharides, disaccharides, oligosaccharides, and polysaccharidesfor example. Typically, sugars as used herein target or delivercopolymers to target cells or tissues, or specific cells types andenhance the association of molecules with the target cells. For example,liver hepatocytes contain asialoglycoprotein (ASGP) receptors.Therefore, galactose-containing targeting groups may be used to targethepatocytes. Examples of galactose containing targeting groups include,but are not limited to, galactose or galactose derivatives such as itsprotected analogs, N-acetylgalactosamine or N-acetylgalactosaminederivatives such as its protected analogs, oligosaccharides, andsaccharide clusters such as Tyr-Glu-Glu-(aminohexyl GalNAc)3,lysine-based galactose clusters, and cholane-based galactose clusters.Other examples of sugars include, but are not limited to, mannose andmannose derivatives such as its protected analogs. In some variations, asugar is a multivalent structure comprising two or more sugar moieties(e.g., three or four moieties). In some such multivalent sugarembodiments, each moiety is connected to a common branching point via alinker. An exemplary multivalent sugar is a tri-N-acetylgalactosamine(tri-NAG) structure having three NAG moieties. Tri-NAG structures aregenerally known in the art and are described, for example, in Lee etal., Carbohydrates and Chemistry and Biology (B. Ernst, G. W. Hart, & P.Sinay, Eds., Wiley-WCH: Weinheim, 2000), Vol. 4, p 459 (and referencescited therein); Biessen et al. J. Med. Chem. 38:1538, 1995; Sliedregt etal., J. Med. Chem. 42:609, 1999; Rensen et al., J. Med. Chem. 47:5798,2004; Khorev et al., Bioorg. Med. Chem. 16:5216, 2008. Another exemplarymultivalent sugar is a bis-mannose-6-phosphate (bis-M6P) structurehaving two mannose-6-phosphate moieties (see, e.g., U.S. Pat. No.8,399,657 to Zhu et al.).

As used herein the term “vitamin” refers to Vitamin A (Retinol), VitaminB1 (Thiamine), Vitamin C (Ascorbic acid), Vitamin D (Calciferol),Vitamin B2 (Riboflavin), Vitamin E (Tocopherol), Vitamin B12(Cobalamins), Vitamin K1 (Phylloquinone), Vitamin B5 (Pantothenic acid),Vitamin B7 (Biotin), Vitamin B6 (Pyridoxine), Vitamin B3 (Niacin),Vitamin B9 (Folic acid) and their derivatives for example. Typically,vitamins as used herein target or deliver copolymers to target cells ortissues, or specific cells types and enhance the association ofmolecules with the target cells. An example of a vitamin as used hereinincludes Vitamin B₉, including folic acid, folate and their derivatives.

When a functional group, such as an amine, is termed “protected”, thismeans that the group is in modified form to preclude undesired sidereactions at the protected site. Suitable protecting groups for thecopolymers of the present disclosure will be recognized from the presentapplication taking into account the level of skill in the art, and withreference to standard textbooks, such as Greene, T. W. et al. ProtectiveGroups in Organic Synthesis Wiley, New York (1991). Carboxy groups canbe protected as esters thereof, for example methyl, ethyl, tert-butyl,benzyl, and 4-nitrobenzyl esters. Hydroxy groups can be protected asethers or esters thereof, for example methoxymethyl ethers,tetrahydropyranyl ethers, benzyl ethers, acetates or benzoates. Mercaptogroups can be protected as thioethers or thioesters, for example pyridylthioethers, maleimide thioethers, tert-butyl thioethers, thioacetates orthiobenzoates. Amino groups can be protected as carbamates, such astert-butoxycarbonyl derivatives, or as amides, such as acetamides andbenzamides.

As is well-known in the art, nomenclature of PEG molecular weight canuse the overall molecular weight (including the PEG end groups) or thenumber of repeat units. For example PEG₁₂ is also known as PEG_(0.6kDa)or PEG_(0.6k). PEG₃₆ is also known as PEG_(1.6kDa) or PEG_(1.6k). PEG₄₈is also known as PEG_(2.2kDa) or PEG_(2.2k). A particular form of PEG₄₈is also known as PEG₂₄-amido-PEG₂₄, but has also been generallydescribed as PEG_(2.2kDa) or PEG_(2.2k).

PEGMA₄₋₅ (Poly(ethylene glycol) methyl ether methacrylate, averageMn=300) is also known as PEGMA_(0.3kDA) or PEGMA_(0.3k) or PEGMA₃₀₀,which is the average molecular weight of a mixture of PEGMA₄ and PEGMA₅.Similarly, PEGMA₇₋₉ (Poly(ethylene glycol) methyl ether methacrylate,average Mn=500) is also known as PEGMA_(0.5kDA) or PEGMA_(0.5k) orPEGMA₅₀₀, which is the average molecular weight of a mixture of PEG₇ andPEG₉. Similarly, PEGMA₁₇₋₁₉ (Poly(ethylene glycol) methyl ethermethacrylate, average Mn=1000) is also known as PEGMA_(1kDA) orPEGMA_(1k) or PEGMA₁₀₀₀, which is the average molecular weight of amixture of PEGMA₁₇ and PEGMA₁₉.

As used herein the term “treating” refers to the administration of acopolymer that eliminates, alleviates, inhibits the progression of, orreverses progression of, in part or in whole, any one or more of thepathological hallmarks or symptoms of any one of the diseases anddisorders being treated. Such disease include, but are not limited toliver cancer, for example hepatocellular carcinoma, cholangiocarcinoma,hepatitis, hypercholesterolemia, liver fibrosis, pulmonary fibrosis,haemochromatosis cancers of the breast, ovaries, pancreas, endometrium,lungs, kidneys, colon, brain, or myeloid cells of hematopoietic origin.Other diseases include ornithine transcarbamylase deficiency (OTCD),alpha-1-antitrypsin deficiency (A1ATD), cystic fibrosis (CF) andhyperoxaluria.

The phrase “therapeutically effective” as used herein is intended toqualify the amount of copolymer or pharmaceutical composition, or thecombined amount of active ingredients in the case of combinationtherapy. This amount or combined amount will achieve the goal oftreating the relevant condition.

As used herein the symbols

and

designate a point of attachment of one molecular moiety to another.

The recitation of numerical ranges by endpoints includes all numberssubsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3,3.80, 4, and 5) and any range within that range.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” encompass embodiments having pluralreferents, unless the content clearly dictates otherwise.

The present disclosure provides for block co-polymers of the formula IT1-L1-[A]_(x)-[B]_(y)-Z  Iwhere

-   T1 is absent or a first targeting moiety;-   L1 is absent or a linking moiety;    A, also referred to as block A or the first block, is a first block    that is a polymer formed from monomers comprising formula A2 or a    random copolymer formed from monomers comprising formulae A1, A2 and    A3; A1 and A2; A2, A4, and A5; A2 and A5; or A4 and A5;

where R¹ is H or C₁-C₆ alkyl, R² is O, S, NH, N(C₁-C₆ alkyl), or(OCH₂CH₂)₁₋₁₂₀, and Q is selected from the group consisting of (i)S-S-pyridyl, (ii) S-S-G, (iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where Vis an amide, ester, imine, oxime, thioester, product of a [3+2]cycloaddition, product of a [4+1] cycloaddition, carbonate, carbamate,urea, acetal, ketal, or hydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀,C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation;

where n is 1-120, R³ is H or C₁-C₆ alkyl, R⁴ is S, O, NH or N(C₁-C₆alkyl), R⁵ is O or S and R⁶ is H, C₁-C₆ alkyl, C₁-C₆ alkyl-NH₂, C₁-C₆alkyl-NH(C₁-C₆ alkyl), C₁-C₆ alkyl-N(C₁-C₆ alkyl)₂;

where R⁷ and R¹⁹ are independently H or C₁-C₆ alkyl, R⁸ is S, O, NH orN(C₁-C₆ alkyl), and R⁹ is O or S and R¹¹ is an amine protecting group;

where n is 1-230, R¹⁷ is H or C₁-C₆ alkyl, R¹⁸ is O, S, NH or N(C₁-C₆alkyl), R¹⁹ is O or S, and R²⁹ is OH, NH, H, T2, or C₁-C₆ alkyl, whereT2 is a second targeting moiety;

where R²¹ is H or C₁-C₆ alkyl, R²² is O, NH or N(C₁-C₆ alkyl), R²³ is H,aryl, arylhalide, alkyl, alkyl alcohol;

B is a second block that is a random copolymer formed from monomerscomprising formulae B1, B2, B3 and B4 or B1, B2 and B3

where R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are independently H or C₁-C₆alkyl, R¹⁸ is O, S, NH, N(C₁-C₆ alkyl), or (OCH₂CH₂)₁₋₁₂₀, and Q isselected from the group consisting of (i) S-S-pyridyl, (ii) S-S-G, (iii)(OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide, ester, imine,oxime, thioester, product of a [3+2] cycloaddition, product of a [4+1]cycloaddition, carbonate, carbamate, urea, acetal, ketal, or hydrazone,and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, orthioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation;-   x is 2-20 kDa;-   y is 2-20 kDa;-   Z is H, SH, C(CH₃)₂CN,

where R²⁴ is S—(C₁-C₁₂ alkyl), aryl, arylhalide, O—(C₁-C₁₂ alkyl),NR²⁵R²⁶ where R²⁵ and R²⁶ are independently H, alkyl, aryl, orheteroaryl;

-   the ratio of x to y is from 2:1 to 1:4; and-   designates a point of attachment.

In some embodiments of a copolymer of Formula I above, the monomer offormula A2 is

where n is 1-20, R³ is H or C₁-C₆ alkyl, R⁴ is S, O, NH or N(C₁-C₆alkyl), R⁵ is O or S and R⁶ is H, C₁-C₆ alkyl, C₁-C₆ alkyl-NH₂, C₁-C₆alkyl-NH(C₁-C₆ alkyl), C₁-C₆ alkyl-N(C₁-C₆ alkyl)₂.

In some embodiments of a copolymer of Formula I above, Q is notS-S-pyridyl and G is a cationic peptide, polyamine, or polycation. Insome such variations, an mRNA molecule is complexed to the cationicpeptide, polyamine, or polycation.

In particular variations of a block copolymer of Formula I as above, T1and/or T2 is selected from the group consisting of an antibody, apeptide, a sugar, and a vitamin. In some embodiments, each of T1 and T2is independently selected from an antibody, a peptide, a sugar, and avitamin (i.e., T1 is a first antibody, peptide sugar, or vitamin, and T2is a second antibody, peptide, sugar, or vitamin, where T1 and T2 may bethe same or different). In some such embodiments as above, the blockcopolymer of Formula I is a copolymer of formula VII as describedhereinbelow.

In some embodiments of a block copolymer of Formula I as above where T1and T2 are both present, T1 and T2 are the same. In other embodiments ofa block copolymer of Formula I as above where T1 and T2 are bothpresent, T1 and T2 are different. In certain variations where T1 and T2are both present and are different, T1 and T2 are both capable ofspecifically binding to the same binding partner on the surface of acell. In other variations where T1 and T2 are both present and aredifferent, T1 and T2 are both capable of specifically binding todifferent binding partners on the surface of the same cell, e.g., thesame cell within a target tissue of a subject. In some such embodimentsas above, the block copolymer of Formula I is a copolymer of formula VIIas described hereinbelow.

In some embodiments of block copolymers of Formula I as above, x is2-15, 2-10 kDa, 3-10 kDa, 3-9 kDa, 3-8 kDa, 3-7 kDa, 3-6 kDa, 4-8 kDa,4-7 kDa, or 4-6 kDa. In some embodiments, y is 2-10 kDa, 3-7 kDa, 3-6kDa, 4-6 kDa, 4.5-5.5 kDa, or 3-5 kDa. In more particular variations, xis 2-15 kDa and y is 3-7 kDa; x is 2-15 kDa and y is 3-6 kDa; x is 2-15kDa and y is 4-6 kDa; x is 2-15 kDa and y is 4.5-5.5 kDa; x is 2-15 kDaand y is 3-5 kDa; x is 2-10 kDa and y is 3-7 kDa; x is 2-10 kDa and y is3-6 kDa; x is 2-10 kDa and y is 4-6 kDa; x is 2-10 kDa and y is 4.5-5.5kDa; x is 2-10 and y is 3-5 kDa; x is 3-10 kDa and y is 3-7 kDa; x is3-10 kDa and y is 3-6 kDa; x is 3-10 kDa and y is 4-6 kDa; x is 3-10 kDaand y is 4.5-5.5 kDa; x is 3-10 kDa and y is 3-5 kDa; x is 3-9 kDa and yis 3-7 kDa; x is 3-9 kDa and y is 3-6 kDa; x is 3-9 kDa and y is 4-6kDa; x is 3-9 kDa and y is 4.5-5.5 kDa; x is 3-9 kDa and y is 3-5 kDa; xis 3-8 kDa and y is 3-7 kDa; x is 3-8 kDa and y is 3-6 kDa; x is 3-8 kDaand y is 4-6 kDa; x is 3-8 kDa and y is 4.5-5.5 kDa; x is 3-8 kDa and yis 3-5 kDa; x is 3-7 kDa and y is 3-7 kDa; x is 3-7 kDa and y is 3-6kDa; x is 3-7 kDa and y is 4-6 kDa; x is 3-7 kDa and y is 4.5-5.5 kDa; xis 3-7 kDa and y is 3-5 kDa; x is 3-6 kDa and y is 3-7 kDa; x is 3-6 kDaand y is 3-6 kDa; x is 3-6 kDa and y is 4-6 kDa; x is 3-6 kDa and y is4.5-5.5 kDa; x is 3-6 kDa and y is 3-5 kDa; x is 4-7 kDa and y is 3-7kDa; x is 4-7 kDa and y is 3-6 kDa; x is 4-7 kDa and y is 4-6 kDa; x is4-7 kDa and y is 4.5-5.5 kDa; x is 4-7 kDa and y is 3-5 kDa; x is 4-6kDa and y is 3-7 kDa; x is 4-6 kDa and y is 3-6 kDa; x is 4-6 kDa and yis 4-6 kDa; x is 4-6 kDa and y is 4.5-5.5 kDa; x is 4-6 kDa and y is 3-5kDa.

Examples of block copolymers of Formula I include those where A is afirst block that is a random copolymer formed from monomers of formulaeA1, A2 and A3 as described above. Additional examples of blockcopolymers of Formula I include those where A is a first block that is arandom copolymer formed from monomers comprising formulae A1 and A2 asdescribed above. Additional examples of block copolymers of Formula Iinclude those where A is a first block that is a polymer formed frommonomer A2 as described above. Additional examples of block copolymersof Formula I include those where A is a first block that is a randomcopolymer formed from monomers comprising formulae A2, A4 and A5 asdescribed above. In some such embodiments as above in which A1 isabsent, the block copolymer of Formula I is a copolymer of formula VIIas described hereinbelow.

An example of a monomer of formula A1 is 2-(pyridin-2-yldisulfanyl)ethylmethacrylate. 2-(Pyridin-2-yldisulfanyl)ethyl methacrylate is alsoreferred to herein as PDSMA. Another example of a monomer of formula A1is

where G is an oligonucleotide, cationic peptide, polyamine, orpolycation.

Examples of monomers of formula A2 include those of formula A2a

where n is 1-120. Other examples of monomers of formula A2 include thoseof formula A2a where n is 1-10 or 3-20. Other examples of monomers offormula A2 include those of formula A2a where n is 3-6. Yet otherexamples of monomer of formula A2 include those of formula A2a where nis 7-20. Additional examples of monomers of formula A2 include those offormula A2a where n is 4 or 5, n is 7-9, or n is 17-19.

Examples of monomers of formula A3 include those of formula A3a

where R¹¹ is an amine protecting group. Other examples of monomers offormula A3 include those of formula A3a where R¹¹ istert-butyloxycarbonyl. An example of monomer A3 is2-(2-((tert-Butoxycarbonyl)amino)ethoxy)ethyl methacrylate which is alsoreferred to herein as BPAM.

A particular example of a block copolymer of Formula I is that where themonomer of formula A1 is

where Q is selected from the group consisting of (i) S-S-pyridyl, (ii)S-S-G, (iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide,ester, imine, oxime, thioester, product of a [3+2] cycloaddition,product of a [4+1] cycloaddition, carbonate, carbamate, urea, acetal,ketal, or hydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and

-   (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation,-   the monomer of formula A2 is

the monomer of formula A3 is absent or2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl methacrylate, and

designates a point of attachment.

An additional example of a block copolymer of Formula I is that wherethe monomer of formula A1 is

wherein Q is selected from the group consisting of (i) S-S-pyridyl, (ii)S-S-G, (iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide,ester, imine, oxime, thioester, product of a [3+2] cycloaddition,product of a [4+1] cycloaddition, carbonate, carbamate, urea, acetal,ketal, or hydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), or(viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G;

-   wherein G is an oligonucleotide, cationic peptide, polyamine, or    polycation,-   the monomer of formula A2 is

the monomer of formula A3 is absent or2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl methacrylate, and

designates a point of attachment.

An additional example of a block copolymer of Formula I is that themonomer of formula A1 is absent, the monomer of formula A2 has theformula A2a where n is 1-120 (e.g., wherein n is 1-10, 3-20, or 7-20),and the monomer of formula A3 is absent. In some variations, the monomerof formula A1 is absent, the monomer of formula A2 is

and the monomer of formula A3 is absent. In other variations, themonomer of A1 is absent, the monomer of formula A2 is selected from thegroup consisting of

and the monomer of A3 is absent. In some such embodiments as above, theblock copolymer of Formula I is a copolymer of formula VII as describedhereinbelow.

An example of a monomer of formula B1 is butyl methacrylate. Butylmethacrylate is also referred to herein as BMA.

An example of a monomer of formula B2 is 2-propyl acrylic acid. 2-Propylacrylic acid is also referred to as 2-n-propyl acrylic acid and2-methylenepentanoic acid. 2-Propyl acrylic acid is also referred toherein as PAA.

An example of a monomer of formula B3 is 2-(dimethylamino)ethylmethacrylate. 2-(dimethylamino)ethyl methacrylate is also commonlyreferred to as 2-dimethylaminoethylester, dimethylaminoethylmethacrylate, N,N-diemthylaminoethyl methacrylate and methacrylic acid2-(dimethylamino)ethyl ester. 2-(dimethylamino)ethyl methacrylate acidis also referred to herein as DMAEMA.

An example of a monomer of formula B4 is 2-(pyridin-2-yldisulfanyl)ethylmethacrylate or

where Q is selected from the group consisting of (i) S-S-pyridyl, (ii)S-S-G, (iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide,ester, imine, oxime, thioester, product of a [3+2] cycloaddition,product of a [4+1] cycloaddition, carbonate, carbamate, urea, acetal,ketal, or hydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and(viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation and    designates a point of attachment.

An example of a block copolymer of Formula I is that where the monomerof formula B1 is butyl methacrylate, the monomer of formula B2 is2-propyl acrylic acid, the monomer of formula B3 is2-(dimethylamino)ethyl methacrylate, and the monomer B4 is absent.

An example of a block copolymer of Formula I is that where the monomerof formula B1 is butyl methacrylate, the monomer of formula B2 is2-propyl acrylic acid, the monomer of formula B3 is2-(dimethylamino)ethyl methacrylate, and the monomer B4 is

wherein Q is selected from the group consisting of (i) S-S-pyridyl, (ii)S-S-G, (iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide,ester, imine, oxime, thioester, product of a [3+2] cycloaddition,product of a [4+1] cycloaddition, carbonate, carbamate, urea, acetal,ketal, or hydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and(viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation and    designates a point of attachment. In some such embodiments as above,    the block copolymer of Formula I is a copolymer of formula VII as    described hereinbelow.

Examples of block copolymers of Formula I include those where T1specifically binds to the asialoglycoprotein (ASGP) receptor.Particularly suitable are structures having one or more N-acetylgalactoseamine (NAG) moieties. For example, in some variations, T1 is

and

designates a point of attachment. In some variations where has multipleNAG moieties, T1 is a tri-N-acetylgalactosamine (tri-NAG) structure. Inone such embodiment, T1 is

and

designates a point of attachment. In some such embodiments as above, theblock copolymer of Formula I is a copolymer of formula VII as describedhereinbelow.

Examples of block copolymers of Formula I include those where L1 is

where m is 1-100 or 10-460 and each of w, x, y, and z is independently1-48. In certain variations of L1 comprising m as above, m is 1-15,10-20, 20-30, 20-25, 11 or 12. In other variations of L1 comprising m asabove, m is 20-60, 25-60, 25-55, 25-50, 25-48, 30-60, 30-55, 30-50,30-48, 34-60, 34-55, 34-50, 34-48, 36-60, 36-55, 36-50, 36-48, 36, or48. In yet other embodiments of L1 comprising m as above, m is 60-460,100-460, 150-460, 200-460, 60-250, 100-250, 150-250, or 200-250. Incertain variations of L1 comprising x and y, x, y and z, or w, x, y andz as above, each of w, x, y, and z is independently 20-30, 20-25, or 23.In other variations of L1 comprising x and y, x, y and z, or w, x, y andz as above, each of w, x, y, and z is independently 1-12, 1-24, 1-36,8-16, 10-14, 20-28, 22-26, 32-40, 34-38, 8-48, 10-48, 20-48, 22-48,32-48, 34-48, or 44-48. In some such embodiments as above, the blockcopolymer of Formula I is a copolymer of formula VII as describedhereinbelow.

Other examples of L1 include—CO—(CH₂CH₂)₁₋₄₆₀—CH₂CH₂NHCO-Ph-C═N—O—(CH₂CH₂)₂₋₂₀—CH₂CH₂NH—CO—CH₂CH₂C(CH₃)(CN)—.Another example of L1 includes—CO—(CH₂CH₂)₁₋₄₆₀—CH₂CH₂NH—CO—CH₂CH₂C(CH₃)(CN)—. Yet another example ofL1 includes—CO—(CH₂CH₂)₁₋₄₆₀—CH₂CH₂NH—CO—(CH₂CH₂)₁₋₅₀—CH₂CH₂NH—CO—CH₂CH₂C(CH₃)(CN)—.In yet other examples, L1 is—CO—(CH₂CH₂)₁₋₄₆₀—CH₂CH₂-x-(CH₂CH₂)₁₋₅₀—CH₂CH₂NH—CO—CH₂CH₂C(CH₃)(CN)—,or —CO—(CH₂CH₂)₁₄₆₀—CH₂CH₂-x -CH₂CH₂C(CH₃)(CN)—, where x is an ester,imine, oxime, thioester, product of a [3+2] cycloaddition, product of a[4+1] cycloaddition, carbonate, carbamate, urea, acetal, ketal, orhydrazine. In some such embodiments as above, the block copolymer ofFormula I is a copolymer of formula VII as described hereinbelow.

Examples of block copolymers of Formula I include those whereT1-L1-together are

wherein m=11 and

designates a point of attachment. In some such embodiments, the blockcopolymer of Formula I is a copolymer of formula VII as describedhereinbelow.

Additional examples of block copolymers of Formula I include those whereT1-L1-together are selected from the group consisting of

and

designates a point of attachment. In some such embodiments, the blockcopolymer of Formula I is a copolymer of formula VII as describedhereinbelow.

Examples of block copolymers of Formula I include those where themonomer of formula A1 is

where Q is selected from the group consisting of (i) S-S-pyridyl, (ii)S-S-G, (iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide,ester, imine, oxime, thioester, product of a [3+2] cycloaddition,product of a [4+1] cycloaddition, carbonate, carbamate, urea, acetal,ketal, or hydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and

-   (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation,-   the monomer of formula A2 is

the monomer of formula A3 is absent or2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl methacrylate, the monomerof formula B1 is butyl methacrylate, the monomer of formula B2 is2-propyl acrylic acid, the monomer of formula B3 is2-(dimethylamino)ethyl methacrylate, and

designates a point of attachment.

Additional examples of block copolymers of Formula I include those wherethe monomer of formula A1 is

where Q is selected from the group consisting of (i) S-S-pyridyl, (ii)S-S-G, (iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide,ester, imine, oxime, thioester, product of a [3+2] cycloaddition,product of a [4+1] cycloaddition, carbonate, carbamate, urea, acetal,ketal, or hydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and

-   (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation,-   the monomer of formula A2 is

the monomer of formula A3 is absent or2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl methacrylate, the monomerof formula B1 is butyl methacrylate, the monomer of formula B2 is2-propyl acrylic acid, the monomer of formula B3 is2-(dimethylamino)ethyl methacrylate, and

designates a point of attachment.

Additional examples of block copolymers of Formula I include those wherethe monomer A1 is absent, the monomer of formula A2 is

the monomer of formula A3 is absent, the monomer of formula B1 is butylmethacrylate, the monomer of formula B2 is 2-propyl acrylic acid, themonomer of formula B3 is 2-(dimethylamino)ethyl methacrylate and themonomer of formula B4 is

where Q is selected from the group consisting of (i) S-S-pyridyl, (ii)S-S-G, (iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide,ester, imine, oxime, thioester, product of a [3+2] cycloaddition,product of a [4+1] cycloaddition, carbonate, carbamate, urea, acetal,ketal, or hydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl),

-   (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation and    designates a point of attachment. In some such embodiments, the    block copolymer of Formula I is a copolymer of formula VII as    described hereinbelow.

Other examples of block copolymers of Formula I include those where themonomer of A1 is absent, the monomer of formula A2 is selected from thegroup consisting of

the monomer of A3 is absent, the monomer of formula B1 is butylmethacrylate, the monomer of formula B2 is 2-propyl acrylic acid, themonomer of formula B3 is 2-(dimethylamino)ethyl methacrylate and themonomer of formula B4 is

where Q is selected from the group consisting of (i) S-S-pyridyl, (ii)S-S-G, (iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide,ester, imine, oxime, thioester, product of a [3+2] cycloaddition,product of a [4+1] cycloaddition, carbonate, carbamate, urea, acetal,ketal, or hydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and

-   (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation and    designates a point of attachment. In some such embodiments as above,    the block copolymer of Formula I is a copolymer of formula VII as    described hereinbelow.

Additional examples of block copolymers of Formula I include those wherethe monomer of formula A1 is

where Q is selected from the group consisting of (i) S-S-pyridyl, (ii)S-S-G, (iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide,ester, imine, oxime, thioester, product of a [3+2] cycloaddition,product of a [4+1] cycloaddition, carbonate, carbamate, urea, acetal,ketal, or hydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and

-   (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation;-   the monomer of formula A2 is

the monomer of formula A3 is absent or2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl methacrylate, the monomerof formula B1 is butyl methacrylate, the monomer of formula B2 is2-propyl acrylic acid, the monomer of formula B3 is2-(dimethylamino)ethyl methacrylate, and T-L-together are

and

designates a point of attachment.

Additional examples of block copolymers of Formula I include those wherethe monomer of formula A1 is

where Q is selected from the group consisting of (i) S-S-pyridyl, (ii)S-S-G, (iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide,ester, imine, oxime, thioester, product of a [3+2] cycloaddition,product of a [4+1] cycloaddition, carbonate, carbamate, urea, acetal,ketal, or hydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and

-   (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation;-   the monomer of formula A2 is

the monomer of formula A3 is absent or2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl methacrylate, the monomerof formula B1 is butyl methacrylate, the monomer of formula B2 is2-propyl acrylic acid, the monomer of formula B3 is2-(dimethylamino)ethyl methacrylate, and T1-L1-together are

and

designates a point of attachment.

Examples of block copolymers of Formula I include those where themonomer of formula A1 is

where Q is selected from the group consisting of (i) S-S-pyridyl, (ii)S-S-G, (iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide,ester, imine, oxime, thioester, product of a [3+2] cycloaddition,product of a [4+1] cycloaddition, carbonate, carbamate, urea, acetal,ketal, or hydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and

-   (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation;-   the monomer of formula A2 is

the monomer of formula A3 is absent or2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl methacrylate, the monomerof formula B1 is butyl methacrylate, the monomer of formula B2 is2-propyl acrylic acid, the monomer of formula B3 is2-(dimethylamino)ethyl methacrylate, x is 2-8 kDa, y is 3-8 kDa, andT1-L1-together are

and

designates a point of attachment.

Examples of block copolymers of Formula I include those where themonomer of formula A1 is

where Q is selected from the group consisting of (i) S-S-pyridyl, (ii)S-S-G, (iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide,ester, imine, oxime, thioester, product of a [3+2] cycloaddition,product of a [4+1] cycloaddition, carbonate, carbamate, urea, acetal,ketal, or hydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and

-   (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   wherein G is an oligonucleotide, cationic peptide, polyamine, or    polycation;-   the monomer of formula A2 is

the monomer of formula A3 is absent or2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl methacrylate, the monomerof formula B1 is butyl methacrylate, the monomer of formula B2 is2-propyl acrylic acid, the monomer of formula B3 is2-(dimethylamino)ethyl methacrylate, x is 2-8 kDa, y is 3-8 kDa, andT1-L1-together are

and

designates a point of attachment.

Examples of block copolymers of Formula I include those of formula IIIT1-L1-[A1-A2]_(x)′-b-[B1-B2-B3]_(y)′Z  IIIwhere the monomer of formula A1 is

and is present in block A in an amount of 5-15 mole percent and whereinQ is selected from the group consisting of (i) S-S-pyridyl, (ii) S-S-G,(iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide, ester,imine, oxime, thioester, product of a [3+2] cycloaddition, product of a[4+1] cycloaddition, carbonate, carbamate, urea, acetal, ketal, orhydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀,C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and

-   (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   wherein G is an oligonucleotide, cationic peptide, polyamine, or    polycation; monomer A2 is

and is present in block A in an amount of 85-95 mole percent; monomer B1is butyl methacrylate and is present in block B in an amount of 53-58mole percent; monomer B2 is 2-propyl acrylic acid and is present inblock B in an amount of 10-15 mole percent; monomer B3 is2-(dimethylamino)ethyl methacrylate and is present in block B in anamount of 30-35 mole percent; x′ is 3-4 kDa; y′ is 5-7 kDa;T1-L1-together are

Z is H, SH,

wherein R²⁴ is S—(C₁-C₁₂ alkyl), aryl, arylhalide, O—(C₁-C₁₂ alkyl),NR²⁵R²⁶ wherein R²⁵ and R²⁶ are independently H, alkyl, aryl, orheteroaryl; and

designates a point of attachment.

In some embodiments of a copolymer of Formula III above, Q is notS-S-pyridyl and G is a cationic peptide, polyamine, or polycation. Insome such variations, an mRNA molecule is complexed to the cationicpeptide, polyamine, or polycation.

Examples of block copolymers of Formula I include those of formula IVT1-L1-[A2]_(x)′-b-[B1-B2-B3-B4]_(y)′Z  IVwherein the monomer A2 is

monomer B1 is butyl methacrylate and is present in block B in an amountof 45-60 mole percent; monomer B2 is 2-propyl acrylic acid and ispresent in block B in an amount of 3-15 mole percent; monomer B3 is2-(dimethylamino)ethyl methacrylate and is present in block B in anamount of 25-40 mole percent; monomer of B4 is

and is present in block B in an amount of 2-25 mole percent and whereinQ is selected from the group consisting of (i) S-S-pyridyl, (ii) S-S-G,(iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide, ester,imine, oxime, thioester, product of a [3+2] cycloaddition, product of a[4+1] cycloaddition, carbonate, carbamate, urea, acetal, ketal, orhydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and

-   (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide; x′ is 3-4 kDa; y′    is 5-7 kDa; T1-L1-together are

Z is H, SH,

wherein R²⁴ is S—(C₁-C₁₂ alkyl), aryl, arylhalide, O—(C₁-C₁₂ alkyl),NR²⁵R²⁶ wherein R²⁵ and R²⁶ are independently H, alkyl, aryl, orheteroaryl; and

designates a point of attachment.

In some embodiments of a copolymer of Formula IV above, Q is notS-S-pyridyl and G is a cationic peptide, polyamine, or polycation. Insome such variations, an mRNA molecule is complexed to the cationicpeptide, polyamine, or polycation.

Additional examples of block copolymers of Formula I include those offormula VIT1-L1-[A2]x′-b-[B1-B2-B3-B4]y′Z VIwherein the monomer A2 is selected from the group consisting of

monomer B1 is butyl methacrylate and is present in block B in an amountof 45-60 mole percent; monomer B2 is 2-propyl acrylic acid and ispresent in block B in an amount of 3-15 mole percent; monomer B3 is2-(dimethylamino)ethyl methacrylate and is present in block B in anamount of 25-40 mole percent; monomer of B4 is

and is present in block B in an amount of 2-25 mole percent and whereinQ is selected from the group consisting of (i) S-S-pyridyl, (ii) S-S-G,(iii) (OCH₂CH₂)₁₋₁₂₀-S-S-G, (iv) V-L3-G where V is an amide, ester,imine, oxime, thioester, product of a [3+2] cycloaddition, product of a[4+1] cycloaddition, carbonate, carbamate, urea, acetal, ketal, orhydrazone, and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆alkyl-(OCH₂CH₂)₁₋₅₀, or thioether,

where R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl), and

-   (viii) S-S-L2-G wherein L2 is

where n=1-35 and

designates a point of attachment of L2 to G,

-   where G is an oligonucleotide, cationic peptide, polyamine, or    polycation;-   x′ is 3-10 kDa; y′ is 3-7 kDa;-   T1-L1-together are selected from the group consisting of

Z is H, SH,

wherein R²⁴ is S-(C₁-C₁₂ alkyl), aryl, arylhalide, O—(C₁-C₁₂ alkyl),NR²⁵R²⁶ wherein R²⁵ and R²⁶ are independently H, alkyl, aryl, orheteroaryl; and

designates a point of attachment.

In some embodiments of a copolymer of Formula VI above, Q is notS-S-pyridyl and G is a cationic peptide, polyamine, or polycation. Insome such variations, an mRNA molecule is complexed to the cationicpeptide, polyamine, or polycation.

In some embodiments of a copolymer of Formula VI above, x is 3-9 kDa; xis 3-8 kDa; x is 3-7 kDa; x is 3-6 kDa; x is 4-8 kDa; x is 4-7 kDa; x is4-6 kDa; y is 3-6 kDa; y is 4-6 kDa; y is 4.5-5.5 kDa; y is 3-5 kDa; xis 3-9 kDa and y is 3-6 kDa; x is 3-9 kDa and y is 4-6 kDa; x is 3-9 kDaand y is 4.5-5.5 kDa; x is 3-9 kDa and y is 3-5 kDa; x is 3-8 kDa and yis 3-6 kDa; x is 3-8 kDa and y is 4-6 kDa; x is 3-8 kDa and y is 4.5-5.5kDa; x is 3-8 kDa and y is 3-5 kDa; x is 3-7 kDa and y is 3-6 kDa; x is3-7 kDa and y is 4-6 kDa; x is 3-7 kDa and y is 4.5-5.5 kDa; x is 3-7kDa and y is 3-5 kDa; x is 3-6 kDa and y is 3-6 kDa; x is 3-6 kDa and yis 4-6 kDa; x is 3-6 kDa and y is 4.5-5.5 kDa; x is 3-6 kDa and y is 3-5kDa; x is 4-7 kDa and y is 3-6 kDa; x is 4-7 kDa and y is 4-6 kDa; x is4-7 kDa and y is 4.5-5.5 kDa; x is 4-7 kDa and y is 3-5 kDa; x is 4-6kDa and y is 3-6 kDa; x is 4-6 kDa and y is 4-6 kDa; x is 4-6 kDa and yis 4.5-5.5 kDa; or x is 4-6 kDa and y is 3-5 kDa.

Additional examples of block copolymers of Formula I include those offormula VIIT1-L1-[A]x′-b-[B1-B2-B3-B4]y′Z  VIIwherein

-   T1 is absent or a first targeting moiety;-   L1 is absent or a linking moiety;    A is a first block that is a polymer formed from monomers comprising    formula A2 or a random copolymer formed from monomers comprising    formulae A2 and A3; A2, A4 and A5; A2 and A5; or A4 and A5;

wherein n is 1-120, R³ is H or C₁-C₆ alkyl, R⁴ is S, O, NH or N(C₁-C₆alkyl), R⁵ is O or S and R⁶ is H, C₁-C₆ alkyl, C₁-C₆ alkyl-NH₂, C₁-C₆alkyl-NH(C₁-C₆ alkyl), C₁-C₆ alkyl-N(C₁-C₆ alkyl)₂;

wherein R⁷ and R¹⁰ are independently H or C₁-C₆ alkyl, R⁸ is S, O, NH orN(C₁-C₆ alkyl), and R⁹ is O or S and R^(H) is an amine protecting group;

wherein n is 1-230, R¹⁷ is H or C₁-C₆ alkyl, R¹⁸ is O, S, NH or N(C₁-C₆alkyl), R¹⁹ is O or S, and R²⁹ is OH, NH, H, T2, or C₁-C₆ alkyl, whereT2 is a second targeting moiety;

wherein R²¹ is H or C₁-C₆ alkyl, R²² is O, NH or N(C₁-C₆ alkyl), R²³ isH, aryl, arylhalide, alkyl, alkyl alcohol;

B is a second block that is a random copolymer formed from monomerscomprising formulae B1, B2, B3 and B4 or B1, B2 and B3

wherein R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are independently H or C₁-C₆alkyl, R¹⁸ is O, S, NH, N(C₁-C₆ alkyl), or (OCH₂CH₂)₁₋₁₂₀, and Q isselected from the group consisting of (i) S-S-G, (ii)(OCH₂CH₂)₁₋₁₂₀-S-S-G, (iii) V-L3-G wherein V is an amide, ester, imine,oxime, thioester, product of a [3+2] cycloaddition, product of a [4+1]cycloaddition, carbonate, carbamate, urea, acetal, ketal, or hydrazone,and L3 is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, orthioether,

wherein R²⁹ is C₁-C₆ alkyl, (OCH₂CH₂)₁₋₅₀, C₁-C₆ alkyl-(OCH₂CH₂)₁₋₅₀, O,NH, or N(C₁-C₆ alkyl),

-   (vii) S-S-L2-G wherein L2 is

wherein n=1-35 and

designates a point of attachment of L2 to G, and

-   (viii) S-S-pyridyl,-   wherein G is a cationic peptide, polyamine, or polycation;-   x is 2-20 kDa;-   y is 2-20 kDa;-   Z is H, SH, C(CH₃)₂CN,

wherein R²⁴ is S—(C₁-C₁₂ alkyl), aryl, arylhalide, O—(C₁-C₁₂ alkyl),NR²⁵R²⁶ wherein R²⁵ and R²⁶ are independently H, alkyl, aryl, orheteroaryl;

-   the ratio of x to y is from 2:1 to 1:4; and-   designates a point of attachment.

In some embodiments of a block copolymer of Formula VII above, themonomer of formula A2 is

where n is 1-20, R³ is H or C₁-C₆ alkyl, R⁴ is S, O, NH or N(C₁-C₆alkyl), R⁵ is O or S and R⁶ is H, C₁-C₆ alkyl, C₁-C₆ alkyl-NH₂, C₁-C₆alkyl-NH(C₁-C₆ alkyl), C₁-C₆ alkyl-N(C₁-C₆ alkyl)₂.

In some embodiments of a copolymer of Formula VII above, Q is notS-S-pyridyl. In some such variations, an mRNA molecule is complexed tothe cationic peptide, polyamine, or polycation.

In some embodiments of block copolymers of Formula VII as above, x is2-15, 2-10 kDa, 3-10 kDa, 3-9 kDa, 3-8 kDa, 3-7 kDa, 3-6 kDa, 4-8 kDa,4-7 kDa, or 4-6 kDa. In some embodiments, y is 2-10 kDa, 3-7 kDa, 3-6kDa, 4-6 kDa, 4.5-5.5 kDa, or 3-5 kDa. In more particular variations, xis 2-15 kDa and y is 3-7 kDa; x is 2-15 kDa and y is 3-6 kDa; x is 2-15kDa and y is 4-6 kDa; x is 2-15 kDa and y is 4.5-5.5 kDa; x is 2-15 kDaand y is 3-5 kDa; x is 2-10 kDa and y is 3-7 kDa; x is 2-10 kDa and y is3-6 kDa; x is 2-10 kDa and y is 4-6 kDa; x is 2-10 kDa and y is 4.5-5.5kDa; x is 2-10 and y is 3-5 kDa; x is 3-10 kDa and y is 3-7 kDa; x is3-10 kDa and y is 3-6 kDa; x is 3-10 kDa and y is 4-6 kDa; x is 3-10 kDaand y is 4.5-5.5 kDa; x is 3-10 kDa and y is 3-5 kDa; x is 3-9 kDa and yis 3-7 kDa; x is 3-9 kDa and y is 3-6 kDa; x is 3-9 kDa and y is 4-6kDa; x is 3-9 kDa and y is 4.5-5.5 kDa; x is 3-9 kDa and y is 3-5 kDa; xis 3-8 kDa and y is 3-7 kDa; x is 3-8 kDa and y is 3-6 kDa; x is 3-8 kDaand y is 4-6 kDa; x is 3-8 kDa and y is 4.5-5.5 kDa; x is 3-8 kDa and yis 3-5 kDa; x is 3-7 kDa and y is 3-7 kDa; x is 3-7 kDa and y is 3-6kDa; x is 3-7 kDa and y is 4-6 kDa; x is 3-7 kDa and y is 4.5-5.5 kDa; xis 3-7 kDa and y is 3-5 kDa; x is 3-6 kDa and y is 3-7 kDa; x is 3-6 kDaand y is 3-6 kDa; x is 3-6 kDa and y is 4-6 kDa; x is 3-6 kDa and y is4.5-5.5 kDa; x is 3-6 kDa and y is 3-5 kDa; x is 4-7 kDa and y is 3-7kDa; x is 4-7 kDa and y is 3-6 kDa; x is 4-7 kDa and y is 4-6 kDa; x is4-7 kDa and y is 4.5-5.5 kDa; x is 4-7 kDa and y is 3-5 kDa; x is 4-6kDa and y is 3-7 kDa; x is 4-6 kDa and y is 3-6 kDa; x is 4-6 kDa and yis 4-6 kDa; x is 4-6 kDa and y is 4.5-5.5 kDa; x is 4-6 kDa and y is 3-5kDa.

Examples of block copolymers of Formula I include

In some embodiments, a block copolymer of Formula I is selected from

-   -   NAG-PEG₁₂-[PEGMA (300,        100%)]_(3.45k)-b-[BMA_(47.5%)-PAA_(9.2%)-DMAEMA_(35.8%)-PDSMA_(7.5%)]_(6.6k);    -   NAG-PEG₁₂-[PEGMA500        (100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(50%)-PAA_(8%)-PDSMA_(6%)]_(5.2k);    -   NAG-PEG₃₆-[PEGMA300,        100%]_(3.5k)-b-[BMA_(50%)-PAA_(9%)-DMAEMA_(35%)-PDSMA_(6%)]_(4.9k);    -   NAG-PEG₂₄-amido-PEG₂₄-[PEGMA300,100%]_(3.6k)-b-[BMA_(50%)-PAA_(11%)-DMAEMA_(32%)-PDSMA_(7%)]_(13.8k);    -   NAG-C5-PEG₂₄-amido-PEG₂₄-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k);    -   NAG-C5-PEG5k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA-_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k);    -   ECT-[PEGMA (300, 58%)-NAG-C5-PEG₃₆        (42%)]_(19.9k)-b-[DMAEMA_(31%)-BMA_(49%)-PAA_(12%)-PDSMA_(8%)]_(5.03k);    -   NAG-PEG₁₂-[PEGMA (300, 73%)-NAG-C5-PEG₃₆        (18%)-TFPMA_(5%)]_(11k)-b-[DMAEMA_(36%)-BMA_(46%)-PAA_(10%)-PDSMA_(7%)]_(5.33k);    -   NAG-C5-PEG10k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k);    -   NAG-C5-PEG20k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k);    -   NAG-C5-PEG24-amido-PEG₂₄-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k);    -   NAG-C5-PEG5k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k);    -   NAG-C5-PEG10k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k);    -   NAG-C5-PEG20k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k);    -   NAG-C5-PEG24-amido-PEG₂₄-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA        (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k);    -   NAG-C5-PEG5k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k);    -   NAG-C5-PEG10k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k);    -   NAG-C5-PEG20k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k);    -   NAG-PEG₃₆-[PEGMA (500,        100%)]_(6.19k)-b-[DMAEMA_(31.6%)-BMA_(48.4%)-PAA_(13.1%)-PDSMA_(6.8%)]_(4.3k);    -   NAG-PEG₃₆-[PEGMA (500,        100%)]_(6.19k)-b-[DMAEMA_(30.8%)-BMA_(50.8%)-PAA_(11.6%)-PDSMA_(6.8%)]_(3.5k);    -   NAG-PEG₄₈-[PEGMA (300,        100%)]_(3.8k)-b-[BMA_(49.3%)-PAA_(9%)-DMAEMA_(31.4%)-PDSMA_(9%)]_(6.3k);    -   NAG-PEG₁₂-[PEGMA(500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(50%)-PAA_(8%)-PDSMA_(6%)]_(5.2k);    -   NAG-PEG₃₆-[PEGMA300,        100%]_(3.5k)-b-[BMA_(50%)-PAA_(9%)-DMAEMA_(35%)-PDSMA_(6%)]_(4.9k);    -   Tri-NAG-PEG₁₂-[PEGMA(300,        80%)-PDSMA_(10%)-BPAM_(10%)]_(6.1k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(4.9k);    -   Tri-NAG-PEG₁₂-[PEGMA(300,        80%)-PDSMA_(10%)-BPAM_(10%)]_(6.4k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(3.2k);        and    -   Tri-NAG-PEG₁₂-[PEGMA(300,        80%)-PDSMA_(10%)-BPAM_(10%)]_(6.4k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(4.2k).        Any one of the above block copolymers may be conjugated to a        cationic peptide, polyamine, or polycation. For example, a        cationic peptide comprising a cysteine residue (e.g., a cationic        peptide having the sequence -Cys-(Lys)₁₀-OH (SEQ ID NO:101) or        -Cys-(Lys)₁₀-NH₂ (SEQ ID NO:103) may be conjugated to the PDSMA        monomer through the cysteine thiol to form a disulfide bridge.

As previously discussed herein, transfection agents used in the arttoday, such as peptides, polymers, and lipids of a cationic nature aswell as nano- and microparticles for example, may achieve hightransfection efficiencies in vitro, achieving similar extents oftransfection without toxicity is difficult in vivo in greatly limitstheir use as delivery systems for nucleic acid-based drugs, particularlyRNA based therapeutics such as siRNA and mRNA therapeutics. The blockcopolymers of Formula I as described herein include, e.g., various blocksizes, mole percentages of monomers, and linkers that surprisinglyprovide for efficient modulation of a target gene, for example decreasedor inhibited expression of a target gene or increased expression of atarget gene, while limiting toxicity in vivo. For example, in certainembodiments, block copolymers of Formula I as described herein, wherethe block copolymer includes a cationic peptide, polyamine, orpolycation and is ionically complexed via the cationic peptide,polyamine, or polycation with an mRNA encoding a protein of interest,provide for an increase in the amount of the encoded protein in a targettissue in vivo. The role and influence of block size, block ratio, molepercentage of monomers, linker length, and various combinations of theseelements on the ability of copolymers as described herein to modulateexpression of a target gene and limit in vivo toxicity is surprising anddescribed in more detail in this specification and demonstrated in theexamples contained herein.

In particular embodiments of block copolymers of Formula I comprising acationic peptide, variation of certain parameters was found to influencethe delivery of mRNA complexed with the copolymer. Such parameters mayalso be varied in a block copolymer of Formula I comprising a polyamineor polycation to similarly influence the delivery of mRNA. For example,increasing the length of the linking moiety L (e.g., increasing thelength of an α-end PEG, such as, for example, increasing m w, x, y,and/or z in a linking moiety of the formula

where each of m, w, x, y, and z is independently 1-100 or 10-460) canresult in increased mRNA delivery to a target tissue. Without beingbound to any particular theory, longer linking moieties may enablebetter presentation of a targeting moiety (e.g., T1), resulting ingreater targeting of copolymer/mRNA particles to a molecular targetpresent on the surface of cells within the target tissue. For example,where T1 includes an N-acetylgalactosamine (NAG) residue, increasing thelength of the linking moiety may enable better presentation of NAG,resulting in greater targeting of particles to the asialoglycoproteinreceptor (ASPGR) on hepatocytes. Further, increasing the size of block A(e.g., increasing the length of PEG in monomer A2) can result inincreased expression of the mRNA. Again without being bound to anyparticular theory, increasing the size of block A appears to increasemRNA and particle stability. (See Example 27, showing longer bloodcirculation at 30 minutes post-dose.) In addition, reducing the size ofblock B may result in improved activity. For example, in some variationsof a copolymer of Formula I, the size of block B is 3-5 kDa. Further, inspecific variations where B1 is butyl methacrylate (BMA), B2 is 2-propylacrylic acid (PAA), and B3 is 2-(dimethylamino)ethyl methacrylate(DMAEMA), a 3:2 ratio of BMA to DMAEMA with a small fraction (e.g.,4-15% mole percent) of PAA appears to enhance mRNA expression.

In other variations of block copolymers of Formula I comprising acationic peptide, polyamine, or polycation, increasing the amount of atargeting moiety on the polymer can improve delivery of mRNA to a targettissue. Increasing the amount of a targeting moiety can be achieved, forexample, by incorporating monomer A4 (comprising a targeting moiety T2)into block A of the polymer. Increasing the amount of a targeting moietymay alternatively, or additionally, include increasing the valency ofthe targeting moiety so as to increase, e.g., the avidity of thetargeting moiety for its specific binding partner on the surface of acell. In specific embodiments, (i) an N-acetylgalactosamine (NAG) sugarresidue is incorporated into monomer A4 of block A to increase theamount of NAG on the polymer (such as on a polymer also comprising NAGas T1 on the α end) and/or (ii) a moiety comprising multiple NAG sugarresidues (e.g., three NAG residues) is used at the α end of the polymerto increase avidity for the asialoglycoprotein receptor (ASPGR) onhepatocytes.

In some variations of a block copolymer of Formula I (e.g., a blockcopolymer of Formula VII), L1 is a polymer having a molecular weight offrom 0.5 kDa to 6 kDa and comprising at least 10 ethylene oxide units.For example, in certain embodiments, L1 has a molecular weight of from 2kDa to 3 kDa and comprises at least 36 ethylene oxide units (e.g., L1can have a weight of 2.2 kDa and have 48 ethylene oxide units, such as astructure having the formula

wherein x and y are each 24 and

designates a point of attachment). In other embodiments, L1 has amolecular weight of from 3 kDa to 6 kDa and comprises at least 48ethylene oxide units. In some such embodiments as above, the monomer offormula A2 is

wherein n is 4 or 5 (PEGMA₃₀₀), n is 7-9 (PEGMA₅₀₀), or n is 17-19(PEGMA₁₀₀₀). In any such embodiments as above, T1 may specifically bindto the asialoglycoprotein receptor. For example, in some embodiments, T1comprises one or more N-acetyl galactoseamine (NAG) moieties. In somesuch embodiments, T1 is a tri-NAG structure having three NAG moieties,such as, e.g., a structure having the formula

where

designates a point of attachment.

Examples of block copolymers of Formula I include those where monomer A1is present in block A in an amount of 3-15 or 5-20 mole percent. Otherexamples of block copolymers of Formula I include those where monomer A1is present in block A in an amount of 5-15 mole percent. Additionalexamples of block copolymers of Formula I include those where monomer A1is present in block A in an amount of 3-10 or 5-10 mole percent.Additional examples of block copolymers of Formula I include those wheremonomer A1 is present in block A in an amount of 10-15 mole percent.Additional examples of block copolymers of Formula I include those wheremonomer A1 is absent. As used herein when describing the amount of agiven monomer in terms of its “mole percent,” the mole percent of agiven monomer is the number of moles of a given monomer in the mixtureof monomers that make up the particular block in which the monomer ispresent as a percentage of the total number of moles of monomers in thatparticular block.

Examples of block copolymers of Formula I include those where monomer A2is present in block A in an amount of 10-30, 30-50, 50-70, 70-90, or70-100 mole percent. Other examples of polymers of Formula I includethose where monomer A2 is present in block A in an amount of 70-85 molepercent. Other examples of polymers of Formula I include those wheremonomer A2 is present in block A in an amount of 75-80 mole percent.Additional examples of polymers of Formula I include those where monomerA2 is present in block A in an amount of 85-95 mole percent. Additionalexample of polymer of Formula I include those where monomer A2 ispresent in block A in an amount of 100 mole percent. In some suchembodiments as above, the block copolymer of Formula I is a copolymer offormula VII.

Examples of block copolymers of Formula I include those where monomer A3is present in block A in an amount of 5-20 mole percent. Other examplesof block copolymers of Formula I include those where monomer A3 ispresent in block A in an amount of 10-15 mole percent. Additionalexamples of Polymers of Formula I include those where monomer A3 isabsent. In some such embodiments as above, the block copolymer ofFormula I is a copolymer of formula VII.

Examples of block copolymers of Formula I include those where monomer A4is present in block A in an amount of 10-50 or 50-95 mole percent. Otherexamples of block copolymers of Formula I include those where monomer A4is present in block A in an amount of 10-30 mole percent. Additionalexamples of block copolymers of Formula I include those where monomer A4is absent. In some such embodiments as above, the block copolymer ofFormula I is a copolymer of formula VII.

Examples of block copolymers of Formula I include those where monomer A5is present in block A in an amount of 0.1-30 mole percent. Otherexamples of block copolymers of Formula I include those where monomer A5is present in block A in an amount of 1-20 mole percent. Additionalexamples of block copolymers of Formula I include those where monomer A5is absent. In some such embodiments as above, the block copolymer ofFormula I is a copolymer of formula VII.

Block copolymers of Formula I possessing the desired gene modulationactivity and minimal in vivo toxicity include those where monomer A1 ispresent in block A in an amount of 5-20 mole percent, monomer A2 ispresent in block A in an amount of 70-90 mole percent, and monomer A3 ispresent in block A in an amount of 5-20 mole percent.

Block copolymers of Formula I possessing the desired gene modulationactivity and minimal in vivo toxicity also include those where monomerA1 is present in block A in an amount of 5-15 mole percent, monomer A2is present in block A in an amount of 70-85 mole percent, and monomer A3is present in block A in an amount of 10-15 mole percent.

Additional block copolymers of Formula I possessing the desired genemodulation activity and minimal in vivo toxicity also include thosewhere monomer A1 is present in block A in an amount of 5-10 molepercent, monomer A2 is present in block A in an amount of 75-80 molepercent, and monomer A3 is present in block A in an amount of 10-15 molepercent.

A particular example of a block copolymer of Formula I is that wheremonomer A1 is present in block A in an amount of 5-15 mole percent,monomer A2 is present in block A in an amount of 85-95 mole percent, andmonomer A3 is absent.

Examples of block copolymers of Formula I include those where monomer B1is present in block B in an amount of 35-65 or 50-60 mole percent. Otherexamples of block copolymers of Formula I include those where monomer B1is present in block B in an amount of 45-60, 48-58 or 53-58 molepercent. Additional examples of block copolymers of Formula I includethose where monomer B1 is present in block B in an amount of 45-55 or50-55 mole percent. Further examples of block copolymers of Formula Iinclude those where monomer B1 is present in block B in an amount of 50mole percent. In some such embodiments as above, the block copolymer ofFormula I is a copolymer of formula VII.

Examples of block copolymers of Formula I include those where monomer B2is present in block B in an amount of 3-15, 5-20 or 10-30 mole percent.Other examples of block copolymers of Formula I include those wheremonomer B2 is present in block B in an amount of 10-25 mole percent.Other examples of block copolymers of Formula I include those wheremonomer B2 is present in block B in an amount of 3-12, 5-15, or 10-15mole percent. Additional examples of block copolymers of Formula Iinclude those where monomer B2 is present in block B in an amount of21-28 mole percent. Further examples of block copolymers of Formula Iinclude those monomer B2 is present in block B in an amount of 25 molepercent. Still further examples of block copolymers of Formula I includethose where monomer B2 is present in block B in an amount of 6-12 or7-10 mole percent. In some such embodiments as above, the blockcopolymer of Formula I is a copolymer of formula VII.

Examples of block copolymers of Formula I include those where monomer B3is present in block B in an amount of 15-35 or 25-40 mole percent. Otherexamples of block copolymers of Formula I include those where monomer B3is present in block B in an amount of 25-35 mole percent. Other examplesof block copolymers of Formula I include those where monomer B3 ispresent in block B in an amount of 30-35 or 30-38 mole percent.Additional examples of block copolymers of Formula I include those wheremonomer B3 is present in block B in an amount of 21-28 mole percent.Further examples of block copolymers of Formula I include those wheremonomer B3 is present in block B in an amount of 25 mole percent. Stillfurther examples of copolymers of Formula I include those where monomerB3 is present in block B in an amount of 32-38 or 33-37 mole percent. Insome such embodiments as above, the block copolymer of Formula I is acopolymer of formula VII.

Examples of block copolymers of Formula I include those where monomer B4is present in block B in an amount of 2-25 or 3-15 mole percent. Otherexamples of block copolymers of Formula I include those where monomer B4is present in block B in an amount of 5-15 mole percent. Other examplesof block copolymers of Formula I include those where monomer B4 ispresent in block B in an amount of 5-10 mole percent. Yet other examplesof block copolymers of Formula I include those where monomer B4 ispresent in block B in an amount of 3-10 or 4-8 mole percent. Additionalexamples of block copolymers of Formula I include those monomer B4 isabsent. In some such embodiments as above where B4 is present, the blockcopolymer of Formula I is a copolymer of formula VII.

Additional block copolymers of Formula I include those where monomer B1is present in block B in an amount of 35-65 mole percent, monomer B2 ispresent in block B in an amount of 10-30 mole percent, and monomer B3 ispresent in block B in an amount of 15-35 mole percent.

Additional block copolymers of Formula I include those where monomer B1is present in block B in an amount of 50-60 mole percent, monomer B2 ispresent in block B in an amount of 10-25 mole percent, and monomer B3 ispresent in block B in an amount of 25-35 mole percent.

Additional block copolymers of Formula I include those where monomer B1is present in block B in an amount of 53-58 mole percent, monomer B2 ispresent in block B in an amount of 10-15 mole percent, and monomer B3 ispresent in block B in an amount of 30-35 mole percent.

Additional block copolymers of Formula I include those where monomer B1is present in block B in an amount of 35-65 mole percent, monomer B2 ispresent in block B in an amount of 10-30 mole percent, monomer B3 ispresent in block B in an amount of 15-35 mole percent and monomer B4 ispresent in block B in an amount of 5-20 mole percent. In some suchembodiments as above, the block copolymer of Formula I is a copolymer offormula VII.

Additional block copolymers of Formula I include those where monomer B1is present in block B in an amount of 50-60 mole percent, monomer B2 ispresent in block B in an amount of 10-25 mole percent, monomer B3 ispresent in block B in an amount of 25-35 mole percent and monomer B4 ispresent in block B in an amount of 5-15 mole percent. In some suchembodiments as above, the block copolymer of Formula I is a copolymer offormula VII.

Additional block copolymers of Formula I include those where monomer B1is present in block B in an amount of 53-58 mole percent, monomer B2 ispresent in block B in an amount of 10-15 mole percent, monomer B3 ispresent in block B in an amount of 30-35 mole percent and monomer B4 ispresent in block B in an amount of 5-10 mole percent. In some suchembodiments as above, the block copolymer of Formula I is a copolymer offormula VII.

Additional block copolymers of Formula I include those where monomer B1is present in block B in an amount of 45-60 mole percent, monomer B2 ispresent in block B in an amount of 3-15 mole percent, monomer B3 ispresent in block B in an amount of 25-40 mole percent and monomer B4 ispresent in block B in an amount of 2-25 mole percent. In some suchembodiments, B2 is present in block B in an amount of 3-12 or 5-15percent. In some such embodiments as above, the block copolymer ofFormula I is a copolymer of formula VII.

Additional block copolymers of Formula I include those where monomer B1is present in block B in an amount of 48-58 mole percent, monomer B2 ispresent in block B in an amount of 5-15 mole percent, monomer B3 ispresent in block B in an amount of 28-35 mole percent and monomer B4 ispresent in block B in an amount of 5-10 mole percent. In some suchembodiments, B2 is present in block B in an amount of 5-10 percent. Insome such embodiments as above, the block copolymer of Formula I is acopolymer of formula VII.

Additional block copolymers of Formula I include those where monomer B1is present in block B in an amount of 45-55 mole percent, monomer B2 ispresent in block B in an amount of 3-12 mole percent, monomer B3 ispresent in block B in an amount of 32-38 mole percent and monomer B4 ispresent in block B in an amount of 3-10 mole percent. In some suchembodiments, B2 is present in block B in an amount of 3-6 or 6-12 molepercent. In some such embodiments as above, the block copolymer ofFormula I is a copolymer of formula VII.

Additional block copolymers of Formula I include those where monomer A1is present in block A in an amount of 5-20 mole percent, monomer A2 ispresent in block A in an amount of 70-90 mole percent, monomer A3 ispresent in block A in an amount of 5-20 mole percent, monomer B1 ispresent in block B in an amount of 35-65 mole percent, monomer B2 ispresent in block B in an amount of 10-30 mole percent, and monomer B3 ispresent in block B in an amount of 15-35 mole percent.

Additional block copolymers of Formula I include those where monomer A1is present in block A in an amount of 5-15 mole percent, monomer A2 ispresent in block A in an amount of 70-85 mole percent, monomer A3 ispresent in block A in an amount of 10-15 mole percent, monomer B1 ispresent in block B in an amount of 50-60 mole percent, monomer B2 ispresent in block B in an amount of 10-25 mole percent, and monomer B3 ispresent in block B in an amount of 25-35 mole percent.

Additional block copolymers of Formula I include those where monomer A1is present in block A in an amount of 5-10 mole percent, monomer A2 ispresent in block A in an amount of 75-80 mole percent, monomer A3 ispresent in block A in an amount of 10-15 mole percent, monomer B1 ispresent in block B in an amount of 53-58 mole percent, monomer B2 ispresent in block B in an amount of 10-15 mole percent, and monomer B3 ispresent in block B in an amount of 30-35 mole percent.

Additional block copolymers of Formula I include those where monomer A1is present in block A in an amount of 5-15 mole percent, monomer A2 ispresent in block A in an amount of 85-95 mole percent, monomer A3 isabsent, monomer B1 is present in block B in an amount of 53-58 molepercent, monomer B2 is present in block B in an amount of 10-15 molepercent, and monomer B3 is present in block B in an amount of 30-35 molepercent.

Examples of block copolymers of Formula I include those where x is 2-10kDa or 3-8 kDa. Other examples of block copolymers of Formula I includethose where x is 5-7 kDa or 8-15 kDa. Additional examples of blockcopolymers of Formula I include those where x is 3-5 kDa. Still otherexamples of block copolymers of Formula I include those where x is 3-15kDa, 3-10 kDa, 3-9 kDa, 3-7 kDa, 3-6 kDa, 4-8 kDa, 4-7 kDa, or 4-6 kDa.In some such embodiments as above, the block copolymer of Formula I is acopolymer of formula VII.

Examples of block copolymers of Formula I include those where y is 2-10kDa or 3-7 kDa. Other examples of block copolymers of Formula I includethose where y is 5-7 kDa. Additional examples of block copolymers ofFormula I include those where y is 3-6 kDa, 4-6 kDa, or 4.5-5.5 kDa. Insome such embodiments as above, the block copolymer of Formula I is acopolymer of formula VII.

Examples of block copolymers of Formula I include those where the ratioof x to y is from 0.25:1 to 2:1; from 0.25:1 to 1.75:1; from 0.25:1 to1.5:1; from 0.25:1 to 1.25:1; from 0.5:1 to 1.25:1; from 0.6 to 1.25:1;from 0.7:1 to 1.25:1; from 0.5:1 to 1.3:1; from 0.6 to 1.3:1; or from0.7:1 to 1.3:1. Additional examples of block copolymers of Formula Iinclude those where the ratio of x to y is from 0.6:1 to 0.8:1; from0.65:1 to 0.75:1; from 0.7:1 to 0.75:1; from 1:1 to 1.3:1; or from 1:1to 1:1.25:1. Another example of copolymers of Formula I include thosewhere the ration of x to y is from 1:1 to 1:3. Yet other examples ofblock copolymers of Formula I include those where the ratio of x to y is1:1 or 0.7:1. In some such embodiments as above, the block copolymer ofFormula I is a copolymer of formula VII.

Examples of block copolymers of Formula I include those where Z is atrithiocarbonate moiety such as

for example. Additional examples of block copolymers of Formula Iinclude those where Z is a moiety derived from the cleavage orderivatization of a trithiocarbonate moiety such as a sulfhydryl moietyfor example.

Additional examples of block copolymers of Formula I include those wherethe oligonucleotide of Q in monomer A1 or B4 (i.e., where G is presentand is an oligonucleotide) is an siRNA, an antisense oligonucleotide, adicer substrate, mRNA, a miRNA, an aiRNA or an shRNA. Additionalexamples of block copolymers of Formula I include those where theoligonucleotide of Q in monomer A1 is an siRNA or mRNA. Further examplesof block copolymers of Formula I include those where the oligonucleotideis an siRNA that inhibits expression of the beta-catenin or MET gene.C-Met, also referred to as MET or MNNG HOS transforming gene, is aproto-oncogene implicated in a variety of cancers, including livercancer, lung cancer, breast cancer, thyroid cancer, gastric cancer,ovarian cancer, pancreatic cancer, head and neck cancer, renal cancerand colorectal cancer, as well as sarcomas, hematologic malignancies,melanoma and central nervous system tumors. C-Met encodes thehepatocycte growth factor receptor (HGFR) protein, which can give riseto invasive growth when activated by its ligand, hepatocyte growthfactor (HGF). Beta catenin, also referred to as CTNNB1, has beenimplicated in a number of cancers, including basal cell carcinoma,colorectal cancer, pilomatrixoma, medullablastoma, and ovarian cancer,as well as adenomatous polyposis of the colon. The gene encoding betacatenin may act as an oncogene in some cases. For example, an increasein beta catenin has been observed in people with basal cell carcinomaand can increase proliferation in related tumors. In addition, mutationsin the gene encoding beta catenin have been observed in various cancers.

Specific examples of oligonucleotides include siRNA oligonucleotidesthat inhibit expression of the MET gene include those in Table 1.

TABLE 1 Synthesized MET dsRNAs. SEQ SEQ ID ID NOSense strand sequence (5′-3′) NO Antisense strand sequence (5′-3′)  1acGfaCfaAfaUfgUfgUfgCfgAfuCfdTsdT 27 GfAfuCfgCfaCfaCfaUfuUfgUfcGfudTsdT 2 gcGfcGfuUfgAfcUfuAfuUfcAfuGfdTsdT 28CfAfuGfaAfuAfaGfuCfaAfcGfcGfcdTsdT  3 gcGfcCfgUfgAfuGfaAfuAfuCfgAfdTsdT29 UfCfgAfuAfuUfcAfuCfaCfgGfcGfcdTsdT  4ucGfcCfgAfaAfuAfcGfgUfcCfuAfdTsdT 30 UfAfgGfaCfcGfuAfuUfuCfgGfcGfadTsdT 5 gcCfgAfaAfuAfcGfgUfcCfuAfuGfdTsdT 31CfAfuAfgGfaCfcGfuAfuUfuCfgGfcdTsdT  6 guAfaGfuGfcCfcGfaAfgUfgUfaAfdTsdT32 UfUfaCfaCfuUfcGfgGfcAfcUfuAfcdTsdT  7guGfcAfgUfaUfcCfuCfuGfaCfaGfdTsdT 33 CfUfgUfcAfgAfgGfaUfaCfuGfcAfcdTsdT 8 cuGfgUfgUfcCfcGfgAfuAfuCfaGfdTsdT 34CfUfgAfuAfuCfcGfgGfaCfaCfcAfgdTsdT  9 ucUfaGfuUfgUfcGfaCfaCfcUfaCfdTsdT35 GfUfaGfgUfgUfcGfaCfaAfcUfaGfadTsdT 10auGfgCfuCfuAfgUfuGfuCfgAfcAfdTsdT 36 UfGfuCfgAfcAfaCfuAfgAfgCfcAfudTsdT11 auUfuCfgCfcGfaAfaUfaCfgGfuCfdTsdT 37GfAfcCfgUfaUfuUfcGfgCfgAfaAfudTsdT 12 ggCfuCfuAfgUfuGfuCfgAfcAfcCfdTsdT38 GfGfuGfuCfgAfcAfaCfuAfgAfgCfcdTsdT 13aaCfuGfgUfgUfcCfcGfgAfuAfuCfdTsdT 39 GfAfuAfuCfcGfgGfaCfaCfcAfgUfudTsdT14 guCfaAfuUfcAfgCfgAfaGfuCfcUfdTsdT 40AfGfgAfcUfuCfgCfuGfaAfuUfgAfcdTsdT 15 cuCfuAfgUfuGfuCfgAfcAfcCfuAfdTsdT41 UfAfgGfuGfuCfgAfcAfaCfuAfgAfgdTsdT 16gcGfaUfcGfgAfgGfaAfuGfcCfuGfdTsdT 42 CfAfgGfcAfuUfcCfuCfcGfaUfcGfcdTsdT17 aaAfuAfcGfgUfcCfuAfuGfgCfuGfdTsdT 43CfAfgCfcAfuAfgGfaCfcGfuAfuUfudTsdT 18 uuUfaCfuUfcUfuGfaCfgGfuCfcAfdTsdT44 UfGfgAfcCfgUfcAfaGfaAfgUfaAfadTsdT 19ucAfuGfgGfuCfaAfuUfcAfgCfgAfdTsdT 45 UfCfgCfuGfaAfuUfgAfcCfcAfuGfadTsdT20 ugUfgCfgAfuCfgGfaGfgAfaUfgCfdTsdT 46GfCfaUfuCfcUfcCfgAfuCfgCfaCfadTsdT 21 gcGfcGfcCfgUfgAfuGfaAfuAfuCfdTsdT47 GfAfuAfuUfcAfuCfaCfgGfcGfcGfcdTsdT 22uuUfcGfcCfgAfaAfuAfcGfgUfcCfdTsdT 48 GfGfaCfcGfuAfuUfuCfgGfcGfaAfadTsdT23 ugGfuUfuCfuCfgAfuCfaGfgAfcCfdTsdT 49GfGfuCfcUfgAfuCfgAfgAfaAfcCfadTsdT 24 uuAfuGfcAfcGfgUfcCfcCfaAfuGfdTsdT50 CfAfuUfgGfgGfaCfcGfuGfcAfuAfadTsdT 25cgAfaAfuAfcGfgUfcCfuAfuGfgCfdTsdT 51 GfCfcAfuAfgGfaCfcGfuAfuUfuCfgdTsdT26 ugGfuGfcCfaCfgAfcAfaAfuGfuGfdTsdT 52CfAfcAfuUfuGfuCfgUfgGfcAfcCfadTsdT

In Table 1, letters in capitals represent RNA nucleotides, lower caseletters “c”, “g”, “a” and “u” represent 2′-O-methyl-modifiednucleotides, “s” represents phosphorothioate and “dT” representsdeoxythymidine residues. Upper case letters A, C, G, U followed by “f”indicate 2′-fluoro nucleotides.

In Table 1, a dsRNA pair is shown within a row. For example, the SEQ IDNO:1 sense strand and the SEQ ID NO:27 antisense strand form a dsRNA,the SEQ ID NO:2 sense strand and the SEQ ID NO:28 antisense strand forma dsRNA, SEQ ID NO:3 sense strand and the SEQ ID NO:29 antisense strandform a dsRNA, and so on.

Specific examples of oligonucleotides include siRNA oligonucleotidesthat inhibit expression of the beta-catenin gene include those in Table2.

The dsRNAs synthesized are presented in Table 2 below.

TABLE 2 Synthesized beta-catenin dsRNAs. SEQ SEQ ID ID NOSense strand sequence (5′-3′) NO Antisense strand sequence (5′-3′) 53caGfgGfgUfcCfuCfuGfuGfaAfcUfdTsdT  77 AfGfuUfcAfcAfgAfgGfaCfcCfcUfgdTsdT54 ugCfuCfuUfcGfuCfaUfcUfgAfcCfdTsdT  78GfGfuCfaGfaUfgAfcGfaAfgAfgCfadTsdT 55 gcUfcUfuCfgUfcAfuCfuGfaCfcAfdTsdT 79 UfGfgUfcAfgAfuGfaCfgAfaGfaGfcdTsdT 56ggAfgCfuAfaAfaUfgGfcAfgUfgCfdTsdT  80 GfCfaCfuGfcCfaUfuUfuAfgCfuCfcdTsdT57 ccUfgUfgCfaGfcUfgGfaAfuUfcUfdTsdT  81AfGfaAfuUfcCfaGfcUfgCfaCfaGfgdTsdT 58 agAfgUfaGfcUfgCfaGfgGfgUfcCfdTsdT 82 GfGfaCfcCfcUfgCfaGfcUfaCfuCfudTsdT 59cuGfaCfuAfuCfcAfgUfuGfaUfgGfdTsdT  83 CfCfaUfcAfaCfuGfgAfuAfgUfcAfgdTsdT60 ccAfuUfcCfaUfuGfuUfuGfuGfcAfdTsdT  84UfGfcAfcAfaAfcAfaUfgGfaAfuGfgdTsdT 61 auAfcCfaUfuCfcAfuUfgUfuUfgUfdTsdT 85 AfCfaAfaCfaAfuGfgAfaUfgGfuAfudTsdT 62gcAfgGfgGfuCfcUfcUfgUfgAfaCfdTsdT  86 GfUfuCfaCfaGfaGfgAfcCfcCfuGfcdTsdT63 ccAfgGfaCfcUfcAfuGfgAfuGfgGfdTsdT  87CfCfcAfuCfcAfuGfaGfgUfcCfuGfgdTsdT 64 uaCfcAfuUfcCfaUfuGfuUfuGfuGfdTsdT 88 CfAfcAfaAfcAfaUfgGfaAfuGfgUfadTsdT 65ugUfgAfaCfuUfgCfuCfaGfgAfcAfdTsdT  89 UfGfuCfcUfgAfgCfaAfgUfuCfaCfadTsdT66 ugGfaUfaUfcGfcCfaGfgAfuGfaUfdTsdT  90AfUfcAfuCfcUfgGfcGfaUfaUfcCfadTsdT 67 ugAfcUfaUfcCfaGfuUfgAfuGfgGfdTsdT 91 CfCfcAfuCfaAfcUfgGfaUfaGfuCfadTsdT 68acCfaUfgCfaGfaAfuAfcAfaAfuGfdTsdT  92 CfAfuUfuGfuAfuUfcUfgCfaUfgGfudTsdT69 acUfgUfuGfgAfuUfgAfuUfcGfaAfdTsdT  93UfUfcGfaAfuCfaAfuCfcAfaCfaGfudTsdT 70 cuAfuCfcAfgUfuGfaUfgGfgCfuGfdTsdT 94 CfAfgCfcCfaUfcAfaCfuGfgAfuAfgdTsdT 71gaCfuAfuCfcAfgUfuGfaUfgGfgCfdTsdT  95 GfCfcCfaUfcAfaCfuGfgAfuAfgUfcdTsdT72 gcUfgAfcUfaUfcCfaGfuUfgAfuGfdTsdT  96CfAfuCfaAfcUfgGfaUfaGfuCfaGfcdTsdT 73 aaUfaCfcAfuUfcCfaUfuGfuUfuGfdTsdT 97 CfAfaAfcAfaUfgGfaAfuGfgUfaUfudTsdT 74acCfcUfgGfuGfcUfgAfcUfaUfcCfdTsdT  98 GfGfaUfaGfuCfaGfcAfcCfaGfgGfudTsdT75 ugCfuUfuAfuUfcUfcCfcAfuUfgAfdTsdT  99UfCfaAfuGfgGfaGfaAfuAfaAfgCfadTsdT 76 agGfaGfcUfaAfaAfuGfgCfaGfuGfdTsdT100 CfAfcUfgCfcAfuUfuUfaGfcUfcCfudTsdT

In Table 2, letters in capitals represent RNA nucleotides, lower caseletters “c”, “g”, “a” and “u” represent 2′-O-methyl-modifiednucleotides, “s” represents phosphorothioate and “dT” representsdeoxythymidine residues. Upper case letters A, C, G, U followed by “f”indicate 2′-fluoro nucleotides.

In Table 2, a dsRNA pair is shown within a row. For example, the SEQ IDNO:53 sense strand and the SEQ ID NO:77 antisense strand form a dsRNA,the SEQ ID NO:54 sense strand and the SEQ ID NO:78 antisense strand forma dsRNA, SEQ ID NO:55 sense strand and the SEQ ID NO:79 antisense strandform a dsRNA, and so on.

Further examples of block copolymers of Formula I include those wherethe oligonucleotide is an siRNA that inhibits expression of the MET genewhere the sense strand consists of a nucleotide sequence of SEQ ID NO:1and the antisense region consists of a nucleotide sequence of SEQ IDNO:27.

Further examples of block copolymers of Formula I include those wherethe oligonucleotide is an siRNA that inhibits expression of thebeta-catenin gene where the sense strand consists of a nucleotidesequence of SEQ ID NO:54 and the antisense region consists of anucleotide sequence of SEQ ID NO:78.

Additional examples of block copolymers of Formula I include those thatinclude a cationic peptide such as those amino acid polymers comprising2-100 amino acid monomers whose overall charge is positive. Additionalexamples of block copolymers of Formula I include those where thecationic peptide in monomer A1 or B4 is a peptide that includes 5-30lysine or arginine residues or a combination thereof. Additionalexamples of cationic peptides include a polylysine or polyargininepeptide. Additional examples of cationic peptides include a polylysineor polyarginine of 5-30 residues. Cationic peptides may include acysteine residue, typically at the amino or carboxyl terminus; suchpeptides are particularly suitable for linkage to an A1 or B4 monomerusing the cysteine thiol to form a disulfide bridge. Examples ofcysteine-containing cationic peptides include -Cys-(Lys)₁₀-OH (SEQ IDNO:101), -Cys-(Arg)₁₀-OH (SEQ ID NO:102), -Cys-(Lys)₁₀-NH₂ (SEQ IDNO:103), -Cys-(Arg)₁₀-NH₂ (SEQ ID NO:104), H₂N-(Lys)₁₀-Cys-OH (SEQ IDNO:105), H₂N-(Arg)₁₀-Cys-OH (SEQ ID NO:106), H₂N-(Lys)₁₀-Cys-NH₂ (SEQ IDNO:115), and H₂N-(Arg)₁₀-Cys-NH₂ (SEQ ID NO:116). Peptides useful inpracticing certain embodiments of the present invention can be preparedusing standard peptide synthesis methodologies known to those of skillin the art. Alternatively, peptides useful in practicing certainembodiments of the present invention including those of SEQ ID NO:103,for example, can be purchased from American Peptide Company of SanDiego, Calif. Yet another example of a cationic peptide includes-Cys-(Lys)₁₀-OH (SEQ ID NO:101) and -Cys-(Lys)₁₀-NH₂ (SEQ ID NO:103). Insome such embodiments as above where B4 is present and A1 is absent, theblock copolymer of Formula I is a copolymer of formula VII.

In particular embodiments of a block copolymer of Formula I including acationic peptide, polyamine, or polycation as above, the copolymer isselected from the group consisting of:

-   (a) a block copolymer of Formula I, wherein G is present (i.e., Q is    not S-S-pyridyl) and G is a cationic peptide, polyamine, or    polycation;-   (b) a block copolymer of Formula I as in (a) above, wherein x is    2-15 kDa, and y is 3-6 kDa, 3-7 kDa, 4-6 kDa, or 3-5 kDa;-   (c) a block copolymer of Formula I as in (a) above, wherein A2 has    the formula A2a, where n is 3-20 or 7-20;-   (d) a block copolymer of Formula I as in (a) above, wherein A2 has    the formula A2a, where n is 7-9 or 17-19;-   (e) a block copolymer of Formula I as in (b) above, wherein A2 has    the formula A2a, where n is 3-20 or 7-20;-   (f) a block copolymer of Formula I as in (b) above, wherein A2 has    the formula A2a, where n is 7-9 or 17-19;-   (g) a block copolymer of Formula I as in (a) above, wherein A1 and    A3 are absent, B4 is present, A2 has the formula A2a, where n is    3-20 or 7-20, B1 is butyl methacrylate, B2 is 2-propyl acrylic acid,    B3 is 2-(dimethylamino)ethyl methacrylate, and B4 is

-   (h) a block copolymer of Formula I as in (g) above, wherein x is    2-15 kDa and y is 3-6 kDa, 3-7 kDa, 4-6 kDa, or 3-5 kDa;-   (i) a block copolymer of Formula I as in (a) above, wherein A1 and    A3 are absent, B4 is present, and T1 is

where

designates a point of attachment;

-   (j) a block copolymer of Formula I as in (i) above, wherein x is    2-15 kDa, y is 3-6 kDa, 3-7 kDa, or 4-6 kDa, and A2 has the formula    A2a, where n is 3-20 or 7-20;

(k) a block copolymer of Formula I as in (j) above, wherein B1 is butylmethacrylate, B2 is 2-propyl acrylic acid, B3 is 2-(dimethylamino)ethylmethacrylate, B4 is

-   (l) a block copolymer of Formula I as in (a) above, wherein L1 is

where m is 20-60 or 60-250, each of w, x, y, and z is independently10-48, and

designates a point of attachment;

-   (m) a block copolymer of Formula I as in (1) above, wherein x is    2-15 kDa, y is 3-6 kDa, 3-7 kDa, 4-6 kDa, or 3-5 kDa, and A2 has the    formula A2a, where n is 3-20 or 7-20;-   (n) a block copolymer of Formula I as in (m) above, wherein A1 and    A3 are absent, B4 is present, B1 is butyl methacrylate, B2 is    2-propyl acrylic acid, B3 is 2-(dimethylamino)ethyl methacrylate,    and B4 is

-   (o) a block copolymer of Formula I as in (a) above, wherein T1 is

where

designates a point of attachment, and L1 is

where m is 20-60 or 60-250, each of w, x, y, and z is independently10-48, and

designates a point of attachment;

-   (p) a block copolymer of Formula I as in (o) above, wherein x is    2-15 kDa, y is 3-6 kDa, 3-7 kDa, 4-6 kDa, or 3-5 kDa, and A2 has the    formula A2a, where n is 3-20 or 7-20;-   (q) a block copolymer of Formula I as in (p) above, wherein A1 and    A3 are absent, B4 is present, B1 is butyl methacrylate, B2 is    2-propyl acrylic acid, B3 is 2-(dimethylamino)ethyl methacrylate,    and B4 is

-   (r) a block copolymer of Formula VI, wherein G is present (i.e., Q    is not S-S-pyridyl) and G is a cationic peptide, polyamine, or    polycation;-   (s) a block copolymer of Formula VII, wherein G is present (i.e., Q    is not S-S-pyridyl);-   (t) a block copolymer of Formula I, wherein A2 has the formula A2a,    where n is 7-9 or 17-19, and wherein L1 is a polymer having a    molecular weight of from 2 kDa to 3 kDa and comprising at least 36    ethylene oxide units, or a polymer having a molecular weight of from    3 kDa to 6 kDa and comprising at least 48 ethylene oxide units;-   (u) a block copolymer as in any one of (a), (b), (c), (d), (e), (f),    (g), (h), (l), (m), (n), (r), and (s) above, wherein T1 is a tri-NAG    structure having three NAG moieties;-   (v) a block copolymer as in any one of (a), (b), (c), (d), (e), (f),    (j), (l), (m), (o), and (p) above, wherein A1 and A3 are absent and    B4 is present;-   (w) a block copolymer as in any one of (a), (b), (c), (d), (e), (f),    (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q), (r), (s),    (t), (u), and (v) wherein A4 and A5 are absent;-   (x) block copolymer as in any one of (a), (b), (c), (d), (e), (f),    (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q), (t), (u),    (v), and (w), wherein the block copolymer is a copolymer of Formula    VII;-   (y) a block copolymer as in any one of (a), (b), (c), (d), (e), (f),    (g), (h), (i), (j), (k), (1), (m), (n), (o), (p), (q), (r), (s),    (t), (u), (v), (w), and (x) above, wherein G is the cationic    peptide;-   (z) a block copolymer of Formula VII wherein the copolymer is a    cationic peptide conjugate of a polymer selected from the group    consisting of:    -   NAG-PEG₁₂-[PEGMA (300,        100%)]_(3.45k)-b-[BMA_(47.5%)-PAA_(9.2%)-DMAEMA_(35.8%)-PDSMA_(7.5%)]_(6.6k);    -   NAG-PEG₁₂-[PEGMA500        (100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(50%)-PAA_(8%)-PDSMA_(6%)]_(5.2k);    -   NAG-PEG₃₆-[PEGMA300,        100%]_(3.5k)-b-[BMA_(50%)-PAA_(9%)-DMAEMA_(35%)-PDSMA_(6%)]_(4.9k);    -   NAG-PEG₂₄-amido-PEG₂₄-[PEGMA300,100%]_(3.6k)-b-[BMA_(50%)-PAA_(11%)-DMAEMA_(32%)-PDSMA_(7%)]_(3.8k);    -   NAG-C5-PEG₂₄-amido-PEG₂₄-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k);    -   NAG-C5-PEG5k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA-_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k);    -   ECT-[PEGMA (300, 58%)-NAG-C5-PEG₃₆        (42%)]_(19.9k)-b-[DMAEMA_(31%)-BMA_(49%)-PAA_(12%)-PDSMA_(8%)]_(5.03k);    -   NAG-PEG₁₂-[PEGMA (300, 73%)-NAG-C5-PEG₃₆        (18%)-TFPMA_(5%)]_(11k)-b-[DMAEMA_(36%)-BMA_(46%)-PAA_(10%)-PDSMA_(7%)]_(5.33k);    -   NAG-C5-PEG10k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k);    -   NAG-C5-PEG20k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k);    -   NAG-C5-PEG24-amido-PEG₂₄-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k);    -   NAG-C5-PEG5k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k);    -   NAG-C5-PEG10k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k);    -   NAG-C5-PEG20k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k);    -   NAG-C5-PEG24-amido-PEG₂₄-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA        (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k);    -   NAG-C5-PEG5k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k);    -   NAG-C5-PEG10k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k);    -   NAG-C5-PEG20k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k);    -   NAG-PEG₃₆-[PEGMA (500,        100%)]_(6.19k)-b-[DMAEMA_(31.6%)-BMA_(48.4%)-PAA_(13.1%)-PDSMA_(6.8%)]_(4.3k);    -   NAG-PEG₃₆-[PEGMA (500,        100%)]_(6.19k)-b-[DMAEMA_(30.8%)-BMA_(50.8%)-PAA_(11.6%)-PDSMA_(6.8%)]_(3.5k);    -   NAG-PEG₄₈-[PEGMA (300,        100%)]_(3.8k)-b-[BMA_(49.3%)-PAA_(9%)-DMAEMA_(31.4%)-PDSMA_(9%)]_(6.3k);    -   NAG-PEG₁₂-[PEGMA(500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(50%)-PAA_(8%)-PDSMA_(6%)]_(5.2k);    -   NAG-PEG₃₆-[PEGMA300,        100%]_(3.5k)-b-[BMA_(50%)-PAA_(9%)-DMAEMA_(35%)-PDSMA_(6%)]_(4.9k);    -   Tri-NAG-PEG₁₂-[PEGMA(300,        80%)-PDSMA_(10%)-BPAM_(10%)]_(6.1k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(4.9k);    -   Tri-NAG-PEG₁₂-[PEGMA(300,        80%)-PDSMA_(10%)-BPAM_(10%)]_(6.4k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(3.2k);        and    -   Tri-NAG-PEG₁₂-[PEGMA(300,        80%)-PDSMA_(10%)-BPAM_(10%)]_(6.4k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(4.2k).    -   wherein the cationic peptide has the sequence -Cys-(Lys)₁₀-OH        (SEQ ID NO:101) or -Cys-(Lys)₁₀-NH₂ (SEQ ID NO:103) and is        conjugated to the PDSMA monomer through the cysteine thiol to        form a disulfide bridge.

The copolymers as described herein are effective transfection agents andtherefore therapeutic agents due to their ability to deliver atherapeutic oligonucleotide intercellularly where they can modulateexpression of a target gene. By modulate, inhibit, down-regulate, orknockdown gene expression, it is meant that the expression of the gene,as measured by the level of RNA transcribed from the gene, or the levelof polypeptide, protein or protein subunit translated from the RNA, isdifferent from that observed in the absence of the copolymers describedherein. For example, the level of RNA transcribed from the gene, or thelevel of polypeptide, protein or protein subunit translated from theRNA, is less than that observed in the presence of a control inactivenucleic acid, a nucleic acid with scrambled sequence or withinactivating mismatches, or observed in the absence of the copolymersdescribed herein when gene expression is modulated, down-regulated orknocked down. Alternatively the level of a polypeptide, protein orprotein subunit in a cell is greater than that observed in the absenceof the copolymers described herein.

The copolymers and formulations described herein effectively transportoligonucleotides, such as mRNA, into cells both in vitro and in vivo.Without being bound to any particular theory, the transport ofoligonucleotides by the copolymers described herein typically occurs viaassociation of the copolymer with the cell membrane and subsequentuptake by the endosomes and eventual disruption of the endosomalmembrane and release of the oligonucleotide, oligonucleotide andcopolymer or copolymer to the cytosol. In the endosomes, the copolymers,and therefore oligonucleotides, are separated from the cytosol. As geneexpression and mRNA translation occurs in the cytosol, theoligonucleotides have to exit the endosome and enter the cytosol foreffective modulation of the target gene or effective translation of atransported mRNA. If the oligonucleotides do not exit the endosome andenter the cytosol, either the endosome matures into or fuses with alysosome leading to degradation of its content, or the endosome fuseswith the cell membrane leading to a return of its content into theextracellular medium. Therefore, without being bound to any particulartheory, the copolymers as described herein are effective in deliveringoligonucleotides intracellularly and thereby modulating a target gene orexpressing a transported mRNA due to their ability to escape fromendosomes. The copolymers as described herein may thus be described as“membrane destabilizing polymers” or “membrane disruptive polymers.”Membrane destabilizing polymers or membrane disruptive polymers candirectly or indirectly elicit a change, such as a permeability changefor example, in a cellular membrane structure, such as an endosomalmembrane for example, so as to permit an agent, for example anoligonucleotide or copolymer or both, to pass through such membranestructure. A membrane disruptive polymer can directly or indirectlyelicit lysis of a cellular vesicle or otherwise disrupt a cellularmembrane for example as observed for a substantial fraction of apopulation of cellular membranes. Generally, membrane destabilizing ormembrane disruptive properties of polymers can be assessed by variousmeans. In one non-limiting approach, a change in a cellular membranestructure can be observed by assessment in assays that measure, directlyor indirectly, release of an agent from cellular membranes, such as anendosomal membrane for example, by determining the presence or absenceof such agent, or an activity of such agent, in an environment externalto such membrane. Another non-limiting approach involves measuring redblood cell lysis, such as hemolysis for example, as a surrogate assayfor a cellular membrane of interest. (See, e.g., International PCTPublications WO 99/34831 and WO 2009/140427.) Such assays may be done ata single pH value or over a range of pH values.

The copolymers and formulations as described herein are useful inmethods for the intracellular delivery of biologically activeoligonucleotides, such as an RNA including siRNA and mRNA for example,to target cells, including target cells in vitro, ex vivo, and in vivo.In some embodiments, a method of delivering a biologically activeoligonucleotide, such as an RNA for example, to a target cell includes(a) contacting a block copolymer of Formula I, where G is present and isan oligonucleotide, such as an RNA for example, with a cell where thecopolymer is introduced into an endosomal membrane within the cellthrough endocytosis; and (b) destabilizing the endosomal membrane,whereby the oligonucelotide is delivered to the cytosol of the cell. Inother embodiments, a method of delivering a biologically activeoligonucleotide to a target cell includes (a) contacting a blockcopolymer of Formula I, where G is present and is a cationic peptide,polyamine, or polycation, and where the copolymer is formulated into acomposition comprising the oligonucleotide, with a cell where thecopolymer is introduced into an endosomal membrane within the cellthrough endocytosis; and (b) destabilizing the endosomal membrane,whereby the oligonucleotide is delivered to the cytosol of the cell. Inother embodiments, a method of delivering a biologically active mRNA toa target cell includes (a) contacting a block copolymer of Formula I,where G is present and is a cationic peptide, polyamine, or polycation,and where the copolymer is formulated into a composition comprising themRNA, with a cell where the copolymer is introduced into an endosomalmembrane within the cell through endocytosis; and (b) destabilizing theendosomal membrane, whereby the mRNA is delivered to the cytosol of thecell.

Examples of methods for the intracellular delivery of a biologicallyactive oligonucleotide to a target cell include those where the cell isin a mammalian animal, including, for example, a human, rodent, murine,bovine, canine, feline, sheep, equine, and simian mammal.

Examples of methods for the intracellular delivery of a biologicallyactive oligonucleotide to a target cell include those where theoligonucleotide is an siRNA, an antisense oligonucleotide, a lockednucleic acid, a dicer substrate, mRNA, a miRNA, an aiRNA or an shRNA.Additional examples of methods for the intracellular delivery of abiologically active oligonucleotide to a target cell include those wherethe oligonucleotide is an siRNA or mRNA.

An example of a method for the intracellular delivery of a biologicallyactive oligonucleotide to a target cell includes those where theoligonucleotide is an mRNA encoding a functional erythropoietin,a-galactosidase, LDL receptor, Factor VII, Factor VIII, Factor IX,alpha-L-iduronidase, iduronate sitlfatase, heparin-N-sulfatase,alpha-N-acetylglucosaminidase, galactose 6-suitatase, β-galactosidase,lysosomal acid lipase, ornithine transcarbamylase, alpha-1-antitrypsinor aryisulfatase-A polypeptide.

Copolymers and formulations as described herein are useful in treating adisease or condition associated with defective gene expression and/oractivity in a subject, such as a mammal for example. Methods oftreatment include administering to a mammal in need of treatment of adisease or condition associated with defective gene expression and/oractivity a therapeutically effective amount of a block copolymer ofFormula I including an oligonucleotide that is homologous to and cansilence, for example by cleavage, a gene or that specifies the aminoacid sequence of a protein and is translated during protein synthesis.

In certain embodiments, the disease or condition associated withdefective gene expression is a disease characterized by a deficiency ina functional polypeptide (also referred to herein as a “diseaseassociated with a protein deficiency”). A copolymer of the presentdisclosure, where the copolymer includes a cationic peptide, polyamine,or polycation, may be formulated into a composition comprising amessenger RNA (mRNA) molecule encoding a protein corresponding to agenetic defect that results in a deficiency of the protein. Fortreatment of the disease associated with the protein deficiency, thecopolymer/mRNA formulation is administered to a subject (e.g., mammalsuch as, for example, a mouse, non-human primate, or human) for deliveryof the mRNA to an appropriate target tissue, where the mRNA istranslated during protein synthesis and the encoded protein is producedin an amount sufficient to treat the disease.

An example of a method of treating a disease or condition associatedwith defective gene expression and/or activity in a subject, such as amammal for example, includes administering to a mammal in need thereof atherapeutically effective amount of a block copolymer of Formula I,wherein Q is S-S-oligonucleotide,

and

designates a point of attachment.

An additional example of a method of treating a disease or conditionassociated with defective gene expression includes a method forincreasing the amount of a protein in a cell by contacting the cell withthe pharmaceutical composition comprising (a) a block copolymer ofFormula I wherein G is present and is a cationic peptide, polyamine, orpolycation, (b) an mRNA molecule and (c) a pharmaceutically acceptablediluent or carrier. In one example the cell in the above describedmethod is in vitro. In another example the cell in the above describedmethod is in vivo.

A further example of a method for treating a disease or conditionassociated with defective gene expression includes a method of treatinga subject having a deficiency in a functional polypeptide comprisingadministering to the subject a pharmaceutical composition comprising ablock copolymer of Formula I, wherein G is present and is a cationicpeptide, polyamine, or polycation, and at least one mRNA molecule atleast a portion of which encodes the functional polypeptide wherefollowing administration the expression of the functional polypeptide isgreater than before administration. In some embodiments, the mRNAencodes a functional erythropoietin, alpha-galactosidase A, LDLreceptor, Factor VII, Factor VIII, Factor IX, alpha-L-iduronidase,iduronate-2-sulfatase, heparan-N-sulfatase,alpha-N-acetylglucosaminidase, galactose 6-sulfatase, acidβ-galactosidase, lysosomal acid lipase, ornithine transcarbamylase,alpha-1-antitrypsin, arylsulfatase A, arylsulfatase B, acid ceramidase,acid α-L-fucosidsase, acid β-glucosidase (also known asglucocerebrosidase), galactocerebrosidase, acid α-mannosidase, acidβ-mannosidase, N-acetylgalactosamine-6-sulfate sulfatase, acidsphingomyelinase, acid α-glucosidase, β-hexosaminidase B,acetyl-CoA:α-glucosaminide N-acetyltransferase,N-acetylglucosamine-6-sulfate sulfatase,alpha-N-acetylgalactosaminidase, sialidase, β-glucuronidase, orβ-hexosaminidase A. In other embodiments, the mRNA encodes a functionalRetinoblastoma protein (pRb), p53 tumor-suppressor protein, Phosphataseand tensin homolog (PTEN), Von Hippel-Lindau tumor suppressor (pVHL),Adenomatous polyposis coli (APC), FAS receptor (FasR), Suppression oftumorigenicity 5 (ST5), YPEL3, Suppressor of tumorigenicity protein 7(ST7), or Suppressor of tumorigenicity 14 protein (ST14). In yet otherembodiments, the mRNA encodes a functional Galactose-1-phosphateuridylyltransferase, Galactokinase, UDP-galactose 4-epimerase,Transthyretin, complement regulatory protein (e.g., factor H, factor I,or membrane cofactor protein), phenylalanine hydroxylase (PAH),homogentisate 1,2-dioxygenase, Porphobilinogen deaminase,hypoxanthine-guanine phosphoribosyltransferase (HGPRT),argininosuccinate lyase (ASL), or P-type ATPase protein, FIC-1.

In an exemplary method for increasing the amount of a protein in a cellby contacting the cell with the pharmaceutical composition comprising(a) a block copolymer of Formula I wherein G is present and is acationic peptide, polyamine, or polycation, (b) an mRNA molecule and (c)a pharmaceutically acceptable diluent or carrier, the mRNA moleculecodes for ornithine transcarbamylase or alpha-1-antitrypsin. In somesuch embodiments, the block copolymer of Formula I is a copolymer offormula VII

In particular embodiments of a composition or method for increasing theamount of a protein in a cell, an mRNA encoding the protein of interestis formulated into a composition comprising a copolymer selected fromthe group consisting of:

-   (a) a block copolymer of Formula I, wherein G is present (i.e., Q is    not S-S-pyridyl) and G is a cationic peptide, polyamine, or    polycation;-   (b) a block copolymer of Formula I as in (a) above, wherein x is    2-15 kDa, and y is 3-6 kDa, 3-7 kDa, 4-6 kDa, or 3-5 kDa;-   (c) a block copolymer of Formula I as in (a) above, wherein A2 has    the formula A2a, where n is 3-20 or 7-20;-   (d) a block copolymer of Formula I as in (a) above, wherein A2 has    the formula A2a, where n is 7-9 or 17-19;-   (e) a block copolymer of Formula I as in (b) above, wherein A2 has    the formula A2a, where n is 3-20 or 7-20;-   (f) a block copolymer of Formula I as in (b) above, wherein A2 has    the formula A2a, where n is 7-9 or 17-19;-   (g) a block copolymer of Formula I as in (a) above, wherein A1 and    A3 are absent, B4 is present, A2 has the formula A2a, where n is    3-20 or 7-20, B1 is butyl methacrylate, B2 is 2-propyl acrylic acid,    B3 is 2-(dimethylamino)ethyl methacrylate, and B4 is

-   (h) a block copolymer of Formula I as in (g) above, wherein x is    2-15 kDa and y is 3-6 kDa, 3-7 kDa, 4-6 kDa, or 3-5 kDa;-   (i) a block copolymer of Formula I as in (a) above, wherein A1 and    A3 are absent, B4 is present, and T1 is

where

designates a point of attachment;

-   (j) a block copolymer of Formula I as in (i) above, wherein x is    2-15 kDa, y is 3-6 kDa, 3-7 kDa, 4-6 kDa, or 3-5 kDa, and A2 has the    formula A2a, where n is 3-20 or 7-20;-   (k) a block copolymer of Formula I as in (j) above, wherein B1 is    butyl methacrylate, B2 is 2-propyl acrylic acid, B3 is    2-(dimethylamino)ethyl methacrylate, B4 is

-   (l) a block copolymer of Formula I as in (a) above, wherein L1 is

where m is 20-60 or 60-250, each of w, x, y, and z is independently10-48, and

designates a point of attachment;

-   (m) a block copolymer of Formula I as in (1) above, wherein x is    2-15 kDa, y is 3-6 kDa, 3-7 kDa, 4-6 kDa, or 3-5 kDa, and A2 has the    formula A2a, where n is 3-20 or 7-20;-   (n) a block copolymer of Formula I as in (m) above, wherein A1 and    A3 are absent, B4 is present, B1 is butyl methacrylate, B2 is    2-propyl acrylic acid, B3 is 2-(dimethylamino)ethyl methacrylate,    and B4 is

-   (o) a block copolymer of Formula I as in (a) above, wherein T1 is

where

designates a point of attachment, and L1 is

where m is 20-60 or 60-250, each of w, x, y, and z is independently10-48, and

designates a point of attachment;

-   (p) a block copolymer of Formula I as in (o) above, wherein x is    2-15 kDa, y is 3-6 kDa, 3-7 kDa, 4-6 kDa, or 3-5 kDa, and A2 has the    formula A2a, where n is 3-20 or 7-20;-   (q) a block copolymer of Formula I as in (p) above, wherein A1 and    A3 are absent, B4 is present, B1 is butyl methacrylate, B2 is    2-propyl acrylic acid, B3 is 2-(dimethylamino)ethyl methacrylate,    and B4 is

-   (r) a block copolymer of Formula VI, wherein G is present (i.e., Q    is not S-S-pyridyl) and G is a cationic peptide, polyamine, or    polycation;-   (s) a block copolymer of Formula VII, wherein G is present (i.e., Q    is not S-S-pyridyl);-   (t) a block copolymer of Formula I, wherein A2 has the formula A2a,    where n is 7-9 or 17-19, and wherein L1 is a polymer having a    molecular weight of from 2 kDa to 3 kDa and comprising at least 36    ethylene oxide units, or a polymer having a molecular weight of from    3 kDa to 6 kDa and comprising at least 48 ethylene oxide units;-   (u) a block copolymer as in any one of (a), (b), (c), (d), (e), (f),    (g), (h), (l), (m), (n), (r), and-   (s) above, wherein T1 is a tri-NAG structure having three NAG    moieties;-   (v) a block copolymer as in any one of (a), (b), (c), (d), (e), (f),    (j), (l), (m), (o), and (p) above, wherein A1 and A3 are absent and    B4 is present;-   (w) a block copolymer as in any one of (a), (b), (c), (d), (e), (f),    (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q), (r), (s),    (t), (u), and (v) wherein A4 and A5 are absent;-   (x) block copolymer as in any one of (a), (b), (c), (d), (e), (f),    (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q), (t), (u),    (v), and (w) wherein the block copolymer is a copolymer of Formula    VII;-   (w) a block copolymer as in any one of (a), (b), (c), (d), (e), (f),    (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q), (r), (s),    (t), (u), (v), (w), and (x) above, wherein G is the cationic    peptide;-   (x) a block copolymer of Formula VII wherein the copolymer is a    cationic peptide conjugate of a polymer selected from the group    consisting of:    -   NAG-PEG₁₂-[PEGMA (300,        100%)]_(3.45k)-b-[BMA_(47.5%)-PAA_(9.2%)-DMAEMA_(35.8%)-PDSMA_(7.5%)]_(6.6k);    -   NAG-PEG₁₂-[PEGMA500        (100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(50%)-PAA_(8%)-PDSMA_(6%)]_(5.2k);    -   NAG-PEG₃₆-[PEGMA300,        100%]_(3.5k)-b-[BMA_(50%)-PAA_(9%)-DMAEMA_(35%)-PDSMA_(6%)]_(4.9k);    -   NAG-PEG₂₄-amido-PEG₂₄-[PEGMA300,100%]_(3.6k)-b-[BMA_(50%)-PAA_(11%)-DMAEMA_(32%)-PDSMA_(7%)]_(3.8k);    -   NAG-C5-PEG₂₄-amido-PEG₂₄-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k);    -   NAG-C5-PEG5k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA-_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k);    -   ECT-[PEGMA (300, 58%)-NAG-C5-PEG₃₆        (42%)]_(19.9k)-b-[DMAEMA_(31%)-BMA_(49%)-PAA_(12%)-PDSMA_(8%)]_(5.03k);    -   NAG-PEG₁₂-[PEGMA (300, 73%)-NAG-C5-PEG₃₆        (18%)-TFPMA_(5%)]_(11k)-b-[DMAEMA_(36%)-BMA_(46%)-PAA_(10%)-PDSMA_(7%)]_(5.33k);    -   NAG-C5-PEG10k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k);    -   NAG-C5-PEG20k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k);    -   NAG-C5-PEG24-amido-PEG₂₄-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k);    -   NAG-C5-PEG5k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k);    -   NAG-C5-PEG10k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k);    -   NAG-C5-PEG20k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k);    -   NAG-C5-PEG24-amido-PEG₂₄-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA        (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k);    -   NAG-C5-PEG5k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k);    -   NAG-C5-PEG10k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k);    -   NAG-C5-PEG20k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k);    -   NAG-PEG₃₆-[PEGMA (500,        100%)]_(6.19k)-b-[DMAEMA_(31.6%)-BMA_(48.4%)-PAA_(13.1%)-PDSMA_(6.8%)]_(4.3k);    -   NAG-PEG₃₆-[PEGMA (500,        100%)]_(6.19k)-b-[DMAEMA_(30.8%)-BMA_(50.8%)-PAA_(11.6%)-PDSMA_(6.8%)]_(3.5k);    -   NAG-PEG₄₈-[PEGMA (300,        100%)]_(3.8k)-b-[BMA_(49.3%)-PAA_(9%)-DMAEMA_(31.4%)-PDSMA_(9%)]_(6.3k);    -   NAG-PEG₁₂-[PEGMA(500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(50%)-PAA_(8%)-PDSMA_(6%)]_(5.2k);    -   NAG-PEG₃₆-[PEGMA300,        100%]_(3.5k)-b-[BMA_(50%)-PAA_(9%)-DMAEMA_(35%)-PDSMA_(6%)]_(4.9k);    -   Tri-NAG-PEG₁₂-[PEGMA(300,        80%)-PDSMA_(10%)-BPAM_(10%)]_(6.1k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(4.9k);    -   Tri-NAG-PEG₁₂-[PEGMA(300,        80%)-PDSMA_(10%)-BPAM_(10%)]_(6.4k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(3.2k);        and    -   Tri-NAG-PEG₁₂-[PEGMA(300,        80%)-PDSMA_(10%)-BPAM_(10%)]_(6.4k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(4.2k).    -   wherein the cationic peptide has the sequence -Cys-(Lys)₁₀-OH        (SEQ ID NO:101) or -Cys-(Lys)₁₀-NH₂ (SEQ ID NO:103) and is        conjugated to the PDSMA monomer through the cysteine thiol to        form a disulfide bridge.

To formulate an mRNA into a composition comprising a copolymer of thepresent disclosure, where the copolymer comprises a cationic peptide,polyamine, or polycation, the copolymer may be solubilized in anaqueous/isotonic buffer at about normal physiological pH (e.g., pH 7.4).Particularly suitable concentrations of solubilized polymer range from 1mg/mL to 50 mg/mL. The mRNA may be prepared using a standard in vitrotranscription reaction according to well-known procedures. The mRNAsolution is typically diluted in an an aqueous/isotonic buffer at aboutnormal physiological pH (e.g., pH 7.4) at a concentration from 0.01mg/mL to 1 mg/mL. The polymer and mRNA stock solutions are then mixedtogether at, e.g., an N:P ratio (nitrogen to phosphorous ratio betweenthe cationic peptide, polyamine, or polycation and the mRNA) rangingfrom 0.5 to 40. After an incubation time, the formulation may be usedfor delivery of the mRNA into target cells (e.g., the formulation may becontacted with cells in vitro or administered to a subject, such asmice, in vivo).

In certain embodiments of a composition or method for increasing theamount of a protein in a cell, the protein is ornithine transcarbamylase(OTC). In such embodiments, an mRNA encoding an OTC protein isformulated into a composition comprising a copolymer of the presentdisclosure such as, for example, a copolymer as set forth in any one of(a)-(z) above. In particular variations, the mRNA molecule encodes anOTC protein comprising an amino acid sequence having at least 90% or atleast 95% sequence identity with residues 35-354 of SEQ ID NO:107 (e.g.,at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%,or 100% sequence identity with residues 35-354 of SEQ ID NO:107). Todirect an encoded OTC protein to the mitochondria of the cell, an mRNAmolecule encoding the OTC protein includes a sequence encoding amitochondrial targeting signal peptide (also referred to herein as a“mitochondrial leader sequence”). The mitochondrial leader sequence maybe that of a native OTC protein (e.g., residues 1-34 of SEQ ID NO:107 (anative human mitochondrial leader sequence) or residues 1-34 of SEQ IDNO:108 (a native mouse mitochondrial leader sequence)), or may bederived from another protein comprising a mitochondrial targeting signalpeptide, or synthesized de novo. An engineered cleavage site may beincluded at the junction between the mitochondrial leader sequence andthe remainder of the polypeptide to optimize proteolytic processing inthe cell. The mitochondrial leader sequence is operably linked to themRNA sequence encoding the mature OTC protein, i.e., the two sequencesare joined in the correct reading frame and positioned to direct thenewly synthesized polypeptide to the mitochondria of a cell.Mitochondrial leader sequences are commonly positioned at the aminoterminus of the protein. In specific variations, the encoded OTC proteinwith a mitochondrial leader sequence has an amino acid sequence as setforth in SEQ ID NO:107 or SEQ ID NO:108. Suitable mRNA sequencesencoding an OTC protein of SEQ ID NO:107, and which may be formulatedinto a composition comprising a copolymer of the present disclosure, maycomprise sequences as shown in SEQ ID NO:112 or SEQ ID NO:114 (codingsequence (CDS) for each corresponding to residues 48-1112). SuitablemRNA sequences encoding an OTC protein of SEQ ID NO:108, and which maybe formulated into a composition comprising a copolymer of the presentdisclosure, may comprise a sequence as shown in SEQ ID NO:113 (codingsequence (CDS) corresponding to residues 48-1112). An OTC-encoding mRNAfor formulation with a copolymer of the present disclosure typicallyfurther includes a poly(A) at its 3′ end (e.g., a polyA tail of about120 adenine residues), which may be added to a construct usingwell-known genetic engineering techniques (e.g., via PCR). Exemplary DNAsequences that may be used for insertion into an appropriate DNA vectorfor production and preparation of mRNA constructs of SEQ ID NOs. 112-114are shown in SEQ ID NOs. 109-111, respectively. Exemplary OTC amino acidsequences and encoding nucleotide sequences are shown in Table 3.

TABLE 3Ornithine Transcarbamylase (OTC) Amino Acid and Encoding NucleotideSequences SEQ ID NO: Sequence Description/Notes 107MLFNLRILLNNAAFRNGHNFMVRNFRCGQPLQNKVQLKGRDL Human ornithineLTLKNFTGEEIKYMLWLSADLKFRIKQKGEYLPLLQGKSLGM transcarbamylaseIFEKRSTRTRLSTETGFALLGGHPCFLTTQDIHLGVNESLTD amino acidTARVLSSMADAVLARVYKQSDLDTLAKEASIPIINGLSDLYH sequence withPIQILADYLTLQEHYSSLKGLTLSWIGDGNNILHSIMMSAAK native (human)FGMHLQAATPKGYEPDASVTKLAEQYAKENGTKLLLTNDPLE mitochondrialAAHGGNVLITDTWISMGQEEEKKKRLQAFQGYQVTMKTAKVA leader sequenceASDWTFLHCLPRKPEEVDDEVFYSPRSLVFPEAENRKWTIMA (leader sequenceVMVSLLTDYSPQLQKPKF underlined) 108MLSNLRILLNNAALRKGHTSVVRHFWCGKPVQSQVQLKGRDL cDNA encodingLTLKNFTGEEIKYMLWLSADLKFRIKQKGEYLPLLQGKSLGM human ornithineIFEKRSTRTRLSTETGFALLGGHPCFLTTQDIHLGVNESLTD transcarabmylaseTARVLSSMADAVLARVYKQSDLDTLAKEASIPIINGLSDLYH with mousePIQILADYLTLQEHYSSLKGLTLSWIGDGNNILHSIMMSAAK mitochondrialFGMHLQAATPKGYEPDASVTKLAEQYAKENGTKLLLTNDPLE leader sequenceAAHGGNVLITDTWISMGQEEEKKKRLQAFQGYQVTMKTAKVA (leader sequenceASDWTFLHCLPRKPEEVDDEVFYSPRSLVFPEAENRKWTIMA underlined)VMVSLLTDYSPQLQKPKF 109 TAATACGACTCACTATAGGG AAATAAGAGAGAAAAGAAGAGTcDNA encoding AAGAAGAAATATAAGAGCCACC ATG CTGTTCAACCTCAGAAThuman ornithine CCTCCTCAATAACGCCGCCTTTAGAAACGGTCATAACTTCATtranscarbamylase, GGTCAGAAACTTTAGATGTGGTCAGCCTCTCCAGAACAAAGTcodon optimized GCAGCTCAAGGGGCGGGACCTGCTCACCCTGAAAAATTTCACfor expression in AGGCGAGGAAATCAAGTACATGCTCTGGCTGTCTGCCGATCT mouseGAAGTTCAGGATCAAGCAGAAGGGCGAATATCTCCCACTGCT (T7 promoterCCAGGGGAAAAGTCTGGGTATGATCTTCGAAAAGCGGAGTAC sequenceTAGGACCAGACTGTCAACAGAGACTGGATTCGCTCTGCTCGG underlined inAGGACACCCATGCTTTCTGACCACACAGGACATTCATCTCGG italics;TGTGAACGAGTCACTGACCGACACAGCTCGAGTCCTCAGCTC start codonCATGGCAGATGCCGTGCTGGCAAGGGTCTACAAACAGAGTGA underlined in bold)CCTCGATACCCTGGCTAAGGAAGCAAGCATCCCCATCATTAATGGACTCTCCGACCTGTATCACCCTATCCAGATTCTGGCCGATTACCTCACCCTGCAGGAGCATTATTCTAGTCTGAAAGGGCTCACACTGAGCTGGATTGGCGACGGAAACAATATCCTGCACTCCATTATGATGTCTGCCGCTAAGTTTGGCATGCATCTGCAGGCAGCCACACCAAAAGGATACGAACCCGATGCTTCCGTGACTAAGCTGGCCGAACAGTATGCTAAAGAGAACGGAACTAAGCTGCTCCTGACCAATGACCCCCTGGAGGCTGCACACGGGGGTAACGTCCTGATCACTGATACCTGGATTTCCATGGGCCAGGAGGAAGAGAAGAAAAAGCGCCTGCAGGCATTCCAGGGATACCAGGTGACAATGAAAACTGCCAAGGTCGCCGCTTCTGATTGGACTTTTCTCCATTGTCTGCCCCGAAAGCCTGAAGAGGTGGACGATGAGGTCTTCTATTCACCTCGGAGCCTGGTGTTTCCAGAAGCCGAGAATCGCAAGTGGACAATCATGGCAGTGATGGTGTCCCTCCTCACAGACTATTCCCCACAGCTCCAGAAGCCCAAGTTTTGAGCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGAGTTTAAACATTTAAAT CT 110 TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGT cDNA encoding AAGAAGAAATATAAGAGCCACC ATGCTCTCTAACCTCAGGAT human ornithineTCTGCTCAACAACGCTGCTCTGCGGAAAGGCCATACCTCTGT transcarbamylaseCGTCAGGCACTTCTGGTGTGGGAAACCCGTGCAGAGCCAGGT with mouseGCAGCTCAAGGGGCGGGACCTGCTCACCCTGAAAAATTTCAC mitochondrialAGGCGAGGAAATCAAGTACATGCTCTGGCTGTCTGCCGATCT leader sequence,GAAGTTCAGGATCAAGCAGAAGGGCGAATATCTCCCACTGCT codon optimizedCCAGGGGAAAAGTCTGGGTATGATCTTCGAAAAGCGGAGTAC for expression inTAGGACCAGACTGTCAACAGAGACTGGATTCGCTCTGCTCGG mouseAGGACACCCATGCTTTCTGACCACACAGGACATTCATCTCGG (T7 promoterTGTGAACGAGTCACTGACCGACACAGCTCGAGTCCTCAGCTC sequenceCATGGCAGATGCCGTGCTGGCAAGGGTCTACAAACAGAGTGA underlined inCCTCGATACCCTGGCTAAGGAAGCAAGCATCCCCATCATTAA italics;TGGACTCTCCGACCTGTATCACCCTATCCAGATTCTGGCCGA start codonTTACCTCACCCTGCAGGAGCATTATTCTAGTCTGAAAGGGCT underlined in bold)CACACTGAGCTGGATTGGCGACGGAAACAATATCCTGCACTCCATTATGATGTCTGCCGCTAAGTTTGGCATGCATCTGCAGGCAGCCACACCAAAAGGATACGAACCCGATGCTTCCGTGACTAAGCTGGCCGAACAGTATGCTAAAGAGAACGGAACTAAGCTGCTCCTGACCAATGACCCCCTGGAGGCTGCACACGGGGGTAACGTCCTGATCACTGATACCTGGATTTCCATGGGCCAGGAGGAAGAGAAGAAAAAGCGCCTGCAGGCATTCCAGGGATACCAGGTGACAATGAAAACTGCCAAGGTCGCCGCTTCTGATTGGACTTTTCTCCATTGTCTGCCCCGAAAGCCTGAAGAGGTGGACGATGAGGTCTTCTATTCACCTCGGAGCCTGGTGTTTCCAGAAGCCGAGAATCGCAAGTGGACAATCATGGCAGTGATGGTGTCCCTCCTCACAGACTATTCCCCACAGCTCCAGAAGCCCAAGTTTTGAGCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGAGTTTAAACATTTAAAT CT 111 TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGT cDNA encoding AAGAAGAAATATAAGAGCCACC ATGCTGTTTAACCTGAGGAT human ornithineTCTGCTGAACAACGCTGCTTTTCGGAACGGCCACAACTTTAT transcarbamylase,GGTGCGGAACTTTCGGTGCGGACAGCCACTGCAGAACAAAGT codon optimizedGCAGCTGAAGGGGAGGGACCTGCTGACCCTGAAAAATTTCAC for expressioninAGGAGAGGAAATCAAGTACATGCTGIGGCTGICTGCCGATCT humanGAAGTTCCGGATCAAGCAGAAGGGCGAATATCTGCCACTGCT (T7 promoterGCAGGGCAAAAGICTGGGGATGATCTTCGAAAAGAGGAGTAC sequenceTCGGACCAGACTGTCAACAGAGACTGGATTCGCTCTGCTGGG underlined inAGGACACCCATGCTTTCTGACCACACAGGACATTCATCTGGG italics;CGTGAACGAGTCACTGACCGACACAGCTCGAGTCCTGAGCTC start codon.CATGGCAGATGCCGTGCTGGCACGGGTCTACAAACAGAGCGA underline in bold)CCTGGATACCCTGGCTAAGGAAGCAAGCATCCCCATCATTAATGGGCTGTCCGACCTGTATCACCCTATCCAGATTCTGGCCGATTACCTGACCCTGCAGGAGCATTATTCTAGTCTGAAAGGCCTGACACTGAGCTGGATTGGGGACGGAAACAATATCCTGCACTCCATTATGATGTCTGCCGCTAAGTTTGGAATGCATCTGCAGGCAGCCACACCAAAAGGCTACGAACCCGATGCCAGTGTGACTAAGCTGGCCGAACAGTATGCTAAAGAGAACGGCACTAAGCTGCTGCTGACCAATGACCCTCTGGAGGCTGCACACGGAGGCAACGTCCTGATCACTGATACCTGGATTTCCATGGGCCAGGAGGAAGAGAAGAAAAAGCGCCTGCAGGCATTCCAGGGGTACCAGGTGACAATGAAAACTGCCAAGGTCGCCGCTTCTGATTGGACTTTTCTGCATTGTCTGCCCCGAAAACCTGAAGAGGTGGACGATGAGGTCTTCTATTCACCTAGGAGCCTGGTGTTTCCAGAAGCCGAGAATCGCAAGTGGACAATCATGGCTGTGATGGTGTCCCTGCTGACTGATTATTCCCCCCAGCTGCAGAAACCTAAGTTCTGAGCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGAGTTTAAACATTTAAAT CT 112GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAG mRNA encoding CCACC AUGCUGUUCAACCUCAGAAUCCUCCUCAAUAACGCCG human ornithineCCUUUAGAAACGGUCAUAACUUCAUGGUCAGAAACUUUAGAU transcarbamylase.GUGGUCAGCCUCUCCAGAACAAAGUGCAGCUCAAGGGGCGGG codon optimizedACCUGCUCACCCUGAAAAAUUUCACAGGCGAGGAAAUCAAGU for expression inACAUGCUCUGGCUGUCUGCCGAUCUGAAGUUCAGGAUCAAGC mouseAGAAGGGCGAAUAUCUCCCACUGCUCCAGGGGAAAAGUCUGG (start codonGUAUGAUCUUCGAAAAGCGGAGUACUAGGACCAGACUGUCAA underlined in bold)CAGAGACUGGAUUCGCUCUGCUCGGAGGACACCCAUGCUUUCUGACCACACAGGACAUUCAUCUCGGUGUGAACGAGUCACUGACCGACACAGCUCGAGUCCUCAGCUCCAUGGCAGAUGCCGUGCUGGCAAGGGUCUACAAACAGAGUGACCUCGAUACCCUGGCUAAGGAAGCAAGCAUCCCCAUCAUUAAUGGACUCUCCGACCUGUAUCACCCUAUCCAGAUUCUGGCCGAUUACCUCACCCUGCAGGAGCAUUAUUCUAGUCUGAAAGGGCUCACACUGAGCUGGAUUGGCGACGGAAACAAUAUCCUGCACUCCAUUAUGAUGUCUGCCGCUAAGUUUGGCAUGCAUCUGCAGGCAGCCACACCAAAAGGAUACGAACCCGAUGCUUCCGUGACUAAGCUGGCCGAACAGUAUGCUAAAGAGAACGGAACUAAGCUGCUCCUGACCAAUGACCCCCUGGAGGCUGCACACGGGGGUAACGUCCUGAUCACUGAUACCUGGAUUUCCAUGGGCCAGGAGGAAGAGAAGAAAAAGCGCCUGCAGGCAUUCCAGGGAUACCAGGUGACAAUGAAAACUGCCAAGGUCGCCGCUUCUGAUUGGACUUUUCUCCAUUGUCUGCCCCGAAAGCCUGAAGAGGUGGACGAUGAGGUCUUCUAUUCACCUCGGAGCCUGGUGUUUCCAGAAGCCGAGAAUCGCAAGUGGACAAUCAUGGCAGUGAUGGUGUCCCUCCUCACAGACUAUUCCCCACAGCUCCAGAAGCCCAAGUUUUGAGCGGCCGCUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGG AAG 113GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAG mRNA encoding CCACC AUGCUCUCUAACCUCAGGAUUCUGCUCAACAACGCUG human ornithineCUCUGCGGAAAGGCCAUACCUCUGUCGUCAGGCACUUCUGGU transcarbamylaseGUGGGAAACCCGUGCAGAGCCAGGUGCAGCUCAAGGGGCGGG with mouseACCUGCUCACCCUGAAAAAUUUCACAGGCGAGGAAAUCAAGU mitochondrialACAUGCUCUGGCUGUCUGCCGAUCUGAAGUUCAGGAUCAAGC leader sequence,AGAAGGGCGAAUAUCUCCCACUGCUCCAGGGGAAAAGUCUGG codon optimizedGUAUGAUCUUCGAAAAGCGGAGUACUAGGACCAGACUGUCAA for expression inCAGAGACUGGAUUCGCUCUGCUCGGAGGACACCCAUGCUUUC mouseUGACCACACAGGACAUUCAUCUCGGUGUGAACGAGUCACUGA (start codonCCGACACAGCUCGAGUCCUCAGCUCCAUGGCAGAUGCCGUGC underlined in bold)UGGCAAGGGUCUACAAACAGAGUGACCUCGAUACCCUGGCUAAGGAAGCAAGCAUCCCCAUCAUUAAUGGACUCUCCGACCUGUAUCACCCUAUCCAGAUUCUGGCCGAUUACCUCACCCUGCAGGAGCAUUAUUCUAGUCUGAAAGGGCUCACACUGAGCUGGAUUGGCGACGGAAACAAUAUCCUGCACUCCAUUAUGAUGUCUGCCGCUAAGUUUGGCAUGCAUCUGCAGGCAGCCACACCAAAAGGAUACGAACCCGAUGCUUCCGUGACUAAGCUGGCCGAACAGUAUGCUAAAGAGAACGGAACUAAGCUGCUCCUGACCAAUGACCCCCUGGAGGCUGCACACGGGGGUAACGUCCUGAUCACUGAUACCUGGAUUUCCAUGGGCCAGGAGGAAGAGAAGAAAAAGCGCCUGCAGGCAUUCCAGGGAUACCAGGUGACAAUGAAAACUGCCAAGGUCGCCGCUUCUGAUUGGACUUUUCUCCAUUGUCUGCCCCGAAAGCCUGAAGAGGUGGACGAUGAGGUCUUCUAUUCACCUCGGAGCCUGGUGUUUCCAGAAGCCGAGAAUCGCAAGUGGACAAUCAUGGCAGUGAUGGUGUCCCUCCUCACAGACUAUUCCCCACAGCUCCAGAAGCCCAAGUUUUGAGCGGCCGCUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGG AAG 114GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAG mRNA encoding CCACC AUGCUGUUUAACCUGAGGAUUCUGCUGAACAACGCUG human ornithineCUUUUCGGAACGGCCACAACUUUAUGGUGCGGAACUUUCGGU transcarbamylase,GCGGACAGCCACUGCAGAACAAAGUGCAGCUGAAGGGGAGGG codon optimizedACCUGCUGACCCUGAAAAAUUUCACAGGAGAGGAAAUCAAGU for expression inACAUGCUGUGGCUGUCUGCCGAUCUGAAGUUCCGGAUCAAGC humanAGAAGGGCGAAUAUCUGCCACUGCUGCAGGGCAAAAGUCUGG (start codonGGAUGAUCUUCGAAAAGAGGAGUACUCGGACCAGACUGUCAA underlined in bold)CAGAGACUGGAUUCGCUCUGCUGGGAGGACACCCAUGCUUUCUGACCACACAGGACAUUCAUCUGGGCGUGAACGAGUCACUGAcCGACACAGCUCGAGUCCUGAGCUCCAUGGCAGAUGCCGUGCUGGCACGGGUCUACAAACAGAGCGACCUGGAUACCCUGGCUAAGGAAGCAAGCAUCCCCAUCAUUAAUGGGCUGUCCGACCUGUAUCACCCUAUCCAGAUUCUGGCCGAUUACCUGACCCUGCAGGAGCAUUAUUCUAGUCUGAAAGGCCUGACACUGAGCUGGAUUGGGGACGGAAACAAUAUCCUGCACUCCAUUAUGAUGUCUGCCGCUAAGUUUGGAAUGCAUCUGCAGGCAGCCACACCAAAAGGCUACGAACCCGAUGCCAGUGUGACUAAGCUGGCCGAACAGUAUGCUAAAGAGAACGGCACUAAGCUGCUGCUGACCAAUGACCCUCUGGAGGCUGCACACGGAGGCAACGUCCUGAUCACUGAUACCUGGAUUUCCAUGGGCCAGGAGGAAGAGAAGAAAAAGCGCCUGCAGGCAUUCCAGGGGUACCAGGUGACAAUGAAAACUGCCAAGGUCGCCGCUUCUGAUUGGACUUUUCUGCAUUGUCUGCCCCGAAAACCUGAAGAGGUGGACGAUGAGGUCUUCUAUUCACCUAGGAGCCUGGUGUUUCCAGAAGCCGAGAAUCGCAAGUGGACAAUCAUGGCUGUGAUGGUGUCCCUGCUGACUGAUUAUUCCCCCCAGCUGCAGAAACCUAAGUUCUGAGCGGCCGCUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGG AAG

In other embodiments of a composition or method for increasing theamount of a protein in a cell, the protein is methylmalonyl CoA mutase(MUT), propionyl CoA carboxylase subunit A (PCCA), propionyl CoAcarboxylase subunit B (PCCB), or a subunit of branched-chain ketoaciddehydrogenase (BCKDH). In such embodiments, an mRNA encoding a MUT,PCCA, PCCB, or BCKDH subunit protein is formulated into a compositioncomprising a copolymer of the present disclosure such as, for example, acopolymer as set forth in any one of (a)-(z) above. In particularvariations, the mRNA molecule encodes a MUT protein comprising an aminoacid sequence having at least 90% or at least 95% sequence identity withresidues 33-750 of SEQ ID NO:117 (e.g., at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% sequence identity withresidues 33-750 of SEQ ID NO:117). In other variations, the mRNAmolecule encodes a PCCA protein comprising an amino acid sequence havingat least 90% or at least 95% sequence identity with residues 53-728 ofSEQ ID NO:119 (e.g., at least 96%, at least 97%, at least 98%, at least99%, at least 99.5%, or 100% sequence identity with residues 53-728 ofSEQ ID NO:119). In other variations, the mRNA molecule encodes a PCCBprotein comprising an amino acid sequence having at least 90% or atleast 95% sequence identity with residues 29-539 of SEQ ID NO:121 (e.g.,at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%,or 100% sequence identity with residues 29-539 of SEQ ID NO:121). Todirect an encoded MUT, PCCA, PCCB, or BCKDH subunit protein to themitochondria of the cell, an mRNA molecule encoding the protein includesa sequence encoding a mitochondrial leader sequence. The mitochondrialleader sequence may be that of a native protein (e.g., residues 1-32 ofSEQ ID NO:117 (a native human MUT mitochondrial leader sequence),residues 1-52 of SEQ ID NO:119 (a native human PCCA mitochondrial leadersequence), or residues 1-28 of SEQ ID NO:121 (a native human PCCBmitochondrial leader sequence)), or may be derived from another proteincomprising a mitochondrial targeting signal peptide, or synthesized denovo. An engineered cleavage site may be included at the junctionbetween the mitochondrial leader sequence and the remainder of thepolypeptide to optimize proteolytic processing in the cell. Themitochondrial leader sequence is operably linked to the mRNA sequenceencoding the mature MUT, PCCA, PCCB, or BCKDH subunit protein, i.e., thetwo sequences are joined in the correct reading frame and positioned todirect the newly synthesized polypeptide to the mitochondria of a cell.In specific variations, the encoded MUT protein with a mitochondrialleader sequence has an amino acid sequence as set forth in SEQ IDNO:117, the encoded PCCA protein with a mitochondrial leader sequencehas an amino acid sequence as set forth in SEQ ID NO:119, or the encodedPCCB protein with a mitochondrial leader sequence has an amino acidsequence as set forth in SEQ ID NO:121. A suitable mRNA sequenceencoding a MUT protein of SEQ ID NO:117, and which may be formulatedinto a composition comprising a copolymer of the present disclosure, maycomprise the sequence shown in SEQ ID NO:118 (coding sequencecorresponding to residues 48-2297). A suitable mRNA sequence encoding aPCCA protein of SEQ ID NO:119, and which may be formulated into acomposition comprising a copolymer of the present disclosure, maycomprise the sequence shown in SEQ ID NO:120 (coding sequencecorresponding to residues 48-2231). A suitable mRNA sequence encoding aPCCB protein of SEQ ID NO:121, and which may be formulated into acomposition comprising a copolymer of the present disclosure, maycomprise the sequence shown in SEQ ID NO:122 (coding sequencecorresponding to residues 48-1664). A MUT-, PCCA-, PCCB-, orBCKDH-subunit-encoding mRNA for formulation with a copolymer of thepresent disclosure typically includes a poly(A) at its 3′ end (e.g., apolyA tail of about 120 adenine residues). Exemplary MUT, PCCA, and PCCBamino acid sequences and encoding nucleotide sequences are shown inTable 4.

TABLE 4 MUT, PCCA, and PCCB Amino Acid and Encoding Nucleotide SequencesSEQ ID NO: Sequence Description/Notes 117MLRAKNQLFLLSPHYLRQVKESSGSRLIQQRLLHQQQPLHPE HumanWAALAKKQLKGKNPEDLIWHTPEGISIKPLYSKRDTMDLPEE methylmalonyl-LPGVKPFTRGPYPTMYTFRPWTIRQYAGFSTVEESNKFYKDN coenzyme AIKAGQQGLSVAFDLATHRGYDSDNPRVRGDVGMAGVAIDTVE mutase amino acidDTKILFDGIPLEKMSVSMTMNGAVIPVLANFIVTGEEQGVPK Sequence withEKLTGTIQNDILKEFMVRNTYIFPPEPSMKIIADIFEYTAKH native (human)MPKFNSISISGYHMQEAGADAILELAYTLADGLEYSRTGLQA mitochondrialGLTIDEFAPRLSFFWGIGMNFYMEIAKMRAGRRLWAHLIEKM leader sequenceFQPKNSKSLLLRAHCQTSGWSLTEQDPYNNIVRTAIEAMAAV (leader sequenceFGGTQSLHTNSFDEALGLPTVKSARIARNTQIIIQEESGIPK underlined)VADPWGGSYMMECLTNDVYDAALKLINEIEEMGGMAKAVAEGIPKLRIEECAARRQARIDSGSEVIVGVNKYQLEKEDAVEVLAIDNTSVRNRQIEKLKKIKSSRDQALAERCLAALTECAASGDGNILALAVDASRARCTVGEITDALKKVFGEHKANDRMVSGAYRQEFGESKEITSAIKRVHKFMEREGRRPRLLVAKMGQDGHDRGAKVIATGFADLGFDVDIGPLFQTPREVAQQAVDADVHAVGISTLAAGHKTLVPELIKELNSLGRPDILVMCGGVIPPQDYEFLFEVGVSNVFGPGTRIPKAAVQVLDDIEKCLEKKQQSV 118GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAG mRNA encoding CCACC AUGUUAAGAGCUAAGAAUCAGCUUUUUUUACUUUCAC humanCUCAUUACCUGAGGCAGGUAAAAGAAUCAUCAGGCUCCAGGC methylmalonyl-UCAUACAGCAACGACUUCUACACCAGCAACAGCCCCUUCACC coenzyme ACAGAAUGGGCUGCCCUGGCUAAAAAGCAGCUGAAAGGCAAAA mutaseACCCAGAAGACCUAAUAUGGCACACCCCGGAAGGGAUCUCUA (start codonUAAAACCCUUGUAUUCCAAGAGAGAUACUAUGGACUUACCUG underlined in bold)AAGAACUUCCAGGAGUGAAGCCAUUCACACGUGGACCAUAUCCUACCAUGUAUACCUUUAGGCCCUGGACCAUCCGCCAGUAUGCUGGUUUUAGUACUGUGGAAGAAAGCAAUAAGUUCUAUAAGGACAACAUUAAGGCUGGUCAGCAGGGAUUAUCAGUUGCCUUUGAUCUGGCGACACAUCGUGGCUAUGAUUCAGACAACCCUCGAGUUCGUGGUGAUGUUGGAAUGGCUGGAGUUGCUAUUGACACUGUGGAAGAUACCAAAAUUCUUUUUGAUGGAAUUCCUUUAGAAAAAAUGUCAGUUUCCAUGACUAUGAAUGGAGCAGUUAUUCCAGUUCUUGCAAAUUUUAUAGUAACUGGAGAAGAACAAGGUGUACCUAAAGAGAAGCUUACUGGUACCAUCCAAAAUGAUAUACUAAAGGAAUUUAUGGUUCGAAAUACAUACAUUUUUCCUCCAGAACCAUCCAUGAAAAUUAUUGCUGACAUAUUUGAAUAUACAGCAAAGCACAUGCCAAAAUUUAAUUCAAUUUCAAUUAGUGGAUACCAUAUGCAGGAAGCAGGGGCUGAUGCCAUUCUGGAGCUGGCCUAUACUUUAGCAGAUGGAUUGGAGUACUCUAGAACUGGACUCCAGGCUGGCCUGACAAUUGAUGAAUUUGCACCAAGGUUGUCUUUCUUCUGGGGAAUUGGAAUGAAUUUCUAUAUGGAAAUAGCAAAGAUGAGAGCUGGUAGAAGACUCUGGGCUCACUUAAUAGAGAAAAUGUUUCAGCCUAAAAACUCAAAAUCUCUUCUUCUAAGAGCACACUGUCAGACAUCUGGAUGGUCACUUACUGAGCAGGAUCCCUACAAUAAUAUUGUCCGUACUGCAAUAGAAGCAAUGGCAGCAGUAUUUGGAGGGACUCAGUCUUUGCACACAAAUUCUUUUGAUGAAGCUUUGGGUUUGCCAACUGUGAAAAGUGCUCGAAUUGCCAGGAACACACAAAUCAUCAUUCAAGAAGAAUCUGGGAUUCCCAAAGUGGCUGAUCCUUGGGGAGGUUCUUACAUGAUGGAAUGUCUCACAAAUGAUGUUUAUGAUGCUGCUUUAAAGCUCAUUAAUGAAAUUGAAGAAAUGGGUGGAAUGGCCAAAGCUGUAGCUGAGGGAAUACCUAAACUUCGAAUUGAAGAAUGUGCUGCCCGAAGACAAGCUAGAAUAGAUUCUGGUUCUGAAGUAAUUGUUGGAGUAAAUAAGUACCAGUUGGAAAAAGAAGACGCUGUAGAAGUUCUGGCAAUUGAUAAUACUUCAGUGCGAAACAGGCAGAUUGAAAAACUUAAGAAGAUCAAAUCCAGCAGGGAUCAAGCUUUGGCUGAACGUUGUCUUGCUGCACUAACCGAAUGUGCUGCUAGCGGAGAUGGAAAUAUCCUGGCUCUUGCAGUGGAUGCAUCUCGGGCAAGAUGUACAGUGGGAGAAAUCACAGAUGCCCUGAAAAAGGUAUUUGGUGAACAUAAAGCGAAUGAUCGAAUGGUGAGUGGAGCAUAUCGCCAGGAAUUUGGAGAAAGUAAAGAGAUAACAUCUGCUAUCAAGAGGGUUCAUAAAUUCAUGGAACGUGAAGGUCGCAGACCUCGUCUUCUUGUAGCAAAAAUGGGACAAGAUGGCCAUGACAGAGGAGCAAAAGUUAUUGCUACAGGAUUUGCUGAUCUUGGUUUUGAUGUGGACAUAGGCCCUCUUUUCCAGACUCCUCGUGAAGUGGCCCAGCAGGCUGUGGAUGCGGAUGUGCAUGCUGUGGGCAUAAGCACCCUCGCUGCUGGUCAUAAAACCCUAGUUCCUGAACUCAUCAAAGAACUUAACUCCCUUGGACGGCCAGAUAUUCUUGUCAUGUGUGGAGGGGUGAUACCACCUCAGGAUUAUGAAUUUCUGUUUGAAGUUGGUGUUUCCAAUGUAUUUGGUCCUGGGACUCGAAUUCCAAAGGCUGCCGUUCAGGUGCUUGAUGAUAUUGAGAAGUGUUUGGAAAAGAAGCAGCAAUCUGUAUAAGCGGCCGCUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAA AGCCUGAGUAGGAAG 119MAGFWVGTAPLVAAGRRGRWPPQQLMLSAALRTLKHVLYYSR Human propionylQCLMVSRNLGSVGYDPNEKTFDKILVANRGEIACRVIRTCKK CoA carboxylase,MGIKTVAIHSDVDASSVHVKMADEAVCVGPAPTSKSYLNMDA alpha polypeptideIMEAIKKTRAQAVHPGYGFLSENKEFARCLAAEDVVFIGPDT (PCCA) aminoHAIQAMGDKIESKLLAKKAEVNTIPGFDGVVKDAEEAVRIAR acid sequence withEIGYPVMIKASAGGGGKGMRIAWDDEETRDGFRLSSQEAASS native (human)FGDDRLLIEKFIDNPRHIEIQVLGDKHGNALWLNERECSIQR mitochondrialRNQKVVEEAPSIFLDAETRRAMGEQAVALARAVKYSSAGTVE leader sequenceFLVDSKKNFYFLEMNTRLQVEHPVTECITGLDLVQEMIRVAK (leader sequenceGYPLRHKQADIRINGWAVECRVYAEDPYKSFGLPSIGRLSQY underlined)QEPLHLPGVRVDSGIQPGSDISIYYDPMISKLITYGSDRTEALKRMADALDNYVIRGVTHNIALLREVIINSRFVKGDISTKFLSDVYPDGFKGHMLTKSEKNQLLAIASSLFVAFQLRAQHFQENSRMPVIKPDIANWELSVKLHDKVHTVVASNNGSVFSVEVDGSKLNVTSTWNLASPLLSVSVDGTQRTVQCLSREAGGNMSIQFLGTVYKVNILTRLAAELNKFMLEKVTEDTSSVLRSPMPGVVVAVSVKPGDAVAEGQEICVIEAMKMQNSMTAGKTGTVKSVHCQA GDTVGEGDLLVELE 120GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAG mRNA encoding CCACC AUGGCGGGGUUCUGGGUCGGGACAGCACCGCUGGUCG human propionylCUGCCGGACGGCGUGGGCGGUGGCCGCCGCAGCAGCUGAUGC CoA carboxylase,UGAGCGCGGCGCUGCGGACCCUGAAGCAUGUUCUGUACUAUU alpha polypeptideCAAGACAGUGCUUAAUGGUGUCCCGUAAUCUUGGUUCAGUGG (pCCA)GAUAUGAUCCUAAUGAAAAAACUUUUGAUAAAAUUCUUGUUG (start codonCUAAUAGAGGAGAAAUUGCAUGUCGGGUUAUUAGAACUUGCA underlined in bold)AGAAGAUGGGCAUUAAGACAGUUGCCAUCCACAGUGAUGUUGAUGCUAGUUCUGUUCAUGUGAAAAUGGCGGAUGAGGCUGUCUGUGUUGGCCCAGCUCCCACCAGUAAAAGCUACCUCAACAUGGAUGCCAUCAUGGAAGCCAUUAAGAAAACCAGGGCCCAAGCUGUACAUCCAGGUUAUGGAUUCCUUUCAGAAAACAAAGAAUUUGCCAGAUGUUUGGCAGCAGAAGAUGUCGUUUUCAUUGGACCUGACACACAUGCUAUUCAAGCCAUGGGCGACAAGAUUGAAAGCAAAUUAUUAGCUAAGAAAGCAGAGGUUAAUACAAUCCCUGGCUUUGAUGGAGUAGUCAAGGAUGCAGAAGAAGCUGUCAGAAUUGCAAGGGAAAUUGGCUACCCUGUCAUGAUCAAGGCCUCAGCAGGUGGUGGUGGGAAAGGCAUGCGCAUUGCUUGGGAUGAUGAAGAGACCAGGGAUGGUUUUAGAUUGUCAUCUCAAGAAGCUGCUUCUAGUUUUGGCGAUGAUAGACUACUAAUAGAAAAAUUUAUUGAUAAUCCUCGUCAUAUAGAAAUCCAGGUUCUAGGUGAUAAACAUGGGAAUGCUUUAUGGCUUAAUGAAAGAGAGUGCUCAAUUCAGAGAAGAAAUCAGAAGGUGGUGGAGGAAGCACCAAGCAUUUUUUUGGAUGCGGAGACUCGAAGAGCGAUGGGAGAACAAGCUGUAGCUCUUGCCAGAGCAGUAAAAUAUUCCUCUGCUGGGACCGUGGAGUUCCUUGUGGACUCUAAGAAGAAUUUUUAUUUCUUGGAAAUGAAUACAAGACUCCAGGUUGAGCAUCCUGUCACAGAAUGCAUUACUGGCCUGGACCUAGUCCAGGAAAUGAUCCGUGUUGCUAAGGGCUACCCUCUCAGGCACAAACAAGCUGAUAUUCGCAUCAACGGCUGGGCAGUUGAAUGUCGGGUUUAUGCUGAGGACCCCUACAAGUCUUUUGGUUUACCAUCUAUUGGGAGAUUGUCUCAGUACCAAGAACCGUUACAUCUACCUGGUGUCCGAGUGGACAGUGGCAUCCAACCAGGAAGUGAUAUUAGCAUUUAUUAUGAUCCUAUGAUUUCAAAACUAAUCACAUAUGGCUCUGAUAGAACUGAGGCACUGAAGAGAAUGGCAGAUGCACUGGAUAACUAUGUUAUUCGAGGUGUUACACAUAAUAUUGCAUUACUUCGAGAGGUGAUAAUCAACUCACGCUUUGUAAAAGGAGACAUCAGCACUAAAUUUCUCUCCGAUGUGUAUCCUGAUGGCUUCAAAGGACACAUGCUAACCAAGAGUGAGAAGAACCAGUUAUUGGCAAUAGCAUCAUCAUUGUUUGUGGCAUUCCAGUUAAGAGCACAACAUUUUCAAGAAAAUUCAAGAAUGCCUGUUAUUAAACCAGACAUAGCCAACUGGGAGCUCUCAGUAAAAUUGCAUGAUAAAGUUCAUACCGUAGUAGCAUCAAACAAUGGGUCAGUGUUCUCGGUGGAAGUUGAUGGGUCGAAACUAAAUGUGACCAGCACGUGGAACCUGGCUUCGCCCUUAUUGUCUGUCAGCGUUGAUGGCACUCAGAGGACUGUCCAGUGUCUUUCUCGAGAAGCAGGUGGAAACAUGAGCAUUCAGUUUCUUGGUACAGUGUACAAGGUGAAUAUCUUAACCAGACUUGCCGCAGAAUUGAACAAAUUUAUGCUGGAAAAAGUGACUGAGGACACAAGCAGUGUUCUGCGUUCCCCGAUGCCCGGAGUGGUGGUGGCCGUCUCUGUCAAGCCUGGAGACGCGGUAGCAGAAGGUCAAGAAAUUUGUGUGAUUGAAGCCAUGAAAAUGCAGAAUAGUAUGACAGCUGGGAAAACUGGCACGGUGAAAUCUGUGCACUGUCAAGCUGGAGACACAGUUGGAGAAGGGGAUCUGCUCGUGGAGCUGGAAUGAGCGGCCGCUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG 121MAAALRVAAVGARLSVLASGLRAAVRSLCSQATSVNERIENK Human propionylRRTALLGGGQRRIDAQHKRGKLTARERISLLLDPGSFVESDM CoA carboxylase,FVEHRCADFGMAADKNKFPGDSVVTGRGRINGRLVYVFSQDF beta polypeptideTVFGGSLSGAHAQKICKIMDQAITVGAPVIGLNDSGGARIQE (PCCB) aminoGVESLAGYADIFLRNVTASGVIPQISLIMGPCAGGAVYSPAL acid sequence withTDFTFMVKDTSYLFITGPDVVKSVTNEDVTQEELGGAKTHTT native (human)MSGVAHRAFENDVDALCNLRDFFNYLPLSSQDPAPVRECHDP mitochondrialSDRLVPELDTIVPLESTKAYNMVDIIHSVVDEREFFEIMPNY leader sequenceAKNIIVGFARMNGRTVGIVGNQPKVASGCLDINSSVKGARFV (leader sequenceRFCDAFNIPLITFVDVPGFLPGTAQEYGGIIRHGAKLLYAFA underlined)EATVPKVTVITRKAYGGAYDVMSSKHLCGDTNYAWPTAEIAVMGAKGAVEIIFKGHENVEAAQAEYIEKFANPFPAAVRGFVDDIIQPSSTRARICCDLDVLASKKVQRPWRKHANIPL 122GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAG mRNA encoding CCACC AUGGCGGCGGCAUUACGGGUGGCGGCGGUCGGGGCAA human propionylGGCUCAGCGUUCUGGCGAGCGGUCUCCGCGCCGCGGUCCGCA CoA carboxylase,GCCUUUGCAGCCAGGCCACCUCUGUUAACGAACGCAUCGAAA beta polypeptideACAAGCGCCGGACCGCGCUGCUGGGAGGGGGCCAACGCCGUA (PCCB)UUGACGCGCAGCACAAGCGAGGAAAGCUAACAGCCAGGGAGA (start codonGGAUCAGUCUCUUGCUGGACCCUGGCAGCUUUGUUGAGAGCG underlined in bold)ACAUGUUUGUGGAACACAGAUGUGCAGAUUUUGGAAUGGCUGCUGAUAAGAAUAAGUUUCCUGGAGACAGCGUGGUCACUGGACGAGGCCGAAUCAAUGGAAGAUUGGUUUAUGUCUUCAGUCAGGAUUUUACAGUUUUUGGAGGCAGUCUGUCAGGAGCACAUGCCCAAAAGAUCUGCAAAAUCAUGGACCAGGCCAUAACGGUGGGGGCUCCAGUGAUUGGGCUGAAUGACUCUGGGGGAGCACGGAUCCAAGAAGGAGUGGAGUCUUUGGCUGGCUAUGCAGACAUCUUUCUGAGGAAUGUUACGGCAUCCGGAGUCAUCCCUCAGAUUUCUCUGAUCAUGGGCCCAUGUGCUGGUGGGGCCGUCUACUCCCCAGCCCUAACAGACUUCACGUUCAUGGUAAAGGACACCUCCUACCUGUUCAUCACUGGCCCUGAUGUUGUGAAGUCUGUCACCAAUGAGGAUGUUACCCAGGAGGAGCUCGGUGGUGCCAAGACCCACACCACCAUGUCAGGUGUGGCCCACAGAGCUUUUGAAAAUGAUGUUGAUGCCUUGUGUAAUCUCCGGGAUUUCUUCAACUACCUGCCCCUGAGCAGUCAGGACCCGGCUCCCGUCCGUGAGUGCCACGAUCCCAGUGACCGUCUGGUUCCUGAGCUUGACACAAUUGUCCCUUUGGAAUCAACCAAAGCCUACAACAUGGUGGACAUCAUACACUCUGUUGUUGAUGAGCGUGAAUUUUUUGAGAUCAUGCCCAAUUAUGCCAAGAACAUCAUUGUUGGUUUUGCAAGAAUGAAUGGGAGGACUGUUGGAAUUGUUGGCAACCAACCUAAGGUGGCCUCAGGAUGCUUGGAUAUUAAUUCAUCUGUGAAAGGGGCUCGUUUUGUCAGAUUCUGUGAUGCAUUCAAUAUUCCACUCAUCACUUUUGUUGAUGUCCCUGGCUUUCUACCUGGCACAGCACAGGAAUACGGGGGCAUCAUCCGGCAUGGUGCCAAGCUUCUCUACGCAUUUGCUGAGGCAACUGUACCCAAAGUCACAGUCAUCACCAGGAAGGCCUAUGGAGGUGCCUAUGAUGUCAUGAGCUCUAAGCACCUUUGUGGUGAUACCAACUAUGCCUGGCCCACCGCAGAGAUUGCAGUCAUGGGAGCAAAGGGCGCUGUGGAGAUCAUCUUCAAAGGGCAUGAGAAUGUGGAAGCUGCUCAGGCAGAGUACAUCGAGAAGUUUGCCAACCCUUUCCCUGCAGCAGUGCGAGGGUUUGUGGAUGACAUCAUCCAACCUUCUUCCACACGUGCCCGAAUCUGCUGUGACCUGGAUGUCUUGGCCAGCAAGAAGGUACAACGUCCUUGGAGAAAACAUGCAAAUAUUCCAUUGUAAGCGGCCGCUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGC CUGAGUAGGAAG

Examples of a disease or condition associated with defective geneexpression and/or activity in a subject treatable by the methodsdisclosed herein include liver cancer, hepatitis, hypercholesterolemia,liver fibrosis or haemochromatosis.

Another example of a disease or condition associated with defective geneexpression and/or activity in a subject treatable by the methodsdisclosed herein includes hepatocellular carcinoma.

Additional examples of a disease or condition associated with defectivegene expression and/or activity in a subject treatable by the methodsdisclosed herein include breast, ovaries, pancreas, endometrium, lungs,kidneys, colon, brain, or myeloid cells of hematopoietic origin.

A further example of a disease or condition associated with defectivegene expression and/or activity in a subject treatable by the methodsdisclosed herein includes glioblastoma.

Further examples of a disease or condition associated with defectivegene expression and/or activity in a subject treatable by the methodsdisclosed herein include ornithine transcarbamylase deficiency (OTCD),alpha-1-antitrypsin deficiency (A1ATD), cystic fibrosis (CF) andhyperoxaluria.

Further examples of a disease or condition associated with defectivegene expression and/or activity in a subject treatable by the methodsdisclosed herein include protein deficiency diseases associated withsingle-gene metabolic defects in the liver. Exemplary protein deficiencydiseases of the liver include diseases associated with urea cycledefects (e.g., ornithine transcarbamylase (OTC) deficiency and carbamoylphosphate synthetase I (CPS1) deficiency); tyrosinemia type 1(fumarylacetoacetase (FAH) enzyme deficiency); primary hyper-oxaluriatype 1 (alanine:glyoxylate-aminotransferase (AGT) deficiency); organicacidemia (e.g., methylmalonic acidemia (MMA; deficiency in, for example,methylmalonyl CoA mutase), propionic acidemia (PA; propionyl CoAcarboxylase (PCC) deficiency), and maple syrup urine disease (MSUD;branched-chain ketoacid dehydrogenase (BCKDH) deficiency)); Wilson'sDisease (deficiency in copper-transporting ATPase, Atp7B);Crigler-Najjar Syndrome Type 1 (bilirubin uridinediphosphateglucuronyltransferase (BGT) enzyme deficiency); hemochromatosis(hepcidin deficiency); glycogen storage disease (GSD) type 1a(glucose-6-phosphatase (G6Pase) deficiency); glycogen storage disease(GSD) type 1b (glucose 6-phosphate translocase deficiency); lysosomalstorage diseases (LSDs; deficiencies in lysosomal enzymes) such as,e.g., Gaucher's Disease types 1, 2, and 3 (lysosomal glucocerebrosidase(GB) deficiency), Niemann-Pick Disease Type C (mutation in either theNPC1 or NPC2 gene), and Niemann-Pick Disease Types A and B (acidsphingomyelinase (ASM) deficiency); alpha-1 antitrypsin (A1AT)deficiency; hemophilia B (Factor IX deficiency); galactosemia types 1,2, and 3 (galactose-1-phosphate uridylyltransferase, galactokinase, andUDP-galactose 4-epimerase deficiencies, respectively);transthyretin-related hereditary amyloidosis (TTR-familial amyloidpolyneuropathy; transthyretin deficiency); atypical haemolytic uremicsyndrome-1 (deficiencies in complement regulatory proteins, e.g., factorH, factor I, or membrane cofactor protein); phenylketonuria(phenylalanine hydroxylase (PAH) deficiency); alcaptonuria(homogentisate 1,2-dioxygenase deficiency); acute intermittent porphyria(porphobilinogen deaminase deficiency); Lesch-Nyhan syndrome(hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency;argininosuccinic aciduria (argininosuccinate lyase (ASL) deficiency);and progressive familial intrahepatic cholestasis (PFIC) (P-type ATPaseprotein, FIC-1 deficiency). Additional examples of protein deficiencydiseases that are lysosomal storage diseases (LSDs) include Fabrydisease (alpha-galactosidase A deficiency); Farber disease (acidceramidase deficiency); fucosidosis (acid α-L-fucosidsase deficiency);GM1 gangliosidosis (acid β-galactosidase deficiency); Hunter syndrome(mucopolysaccharidosis type II (MPS II); iduronate-2-sulfatasedeficiency); Hurler-Scheie, Hurler, and Scheie syndromes(mucopolysaccharidosis type I (MPS I); alpha-L-iduronidase deficiency);Krabbe disease (galactocerebrosidase deficiency); α-mannosidosis (acidα-mannosidase deficiency); β-mannosidosis (acid β-mannosidasedeficiency); Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI (MPSVI); arylsulfatase B deficiency); metachromatic leukodystrophy(arylsulfatase A deficiency); Morquio syndrome type A(mucopolysaccharidosis type IVA (MPS IVA);N-acetylgalactosamine-6-sulfate sulfatase deficiency); Morquio syndrometype B (mucopolysaccharidosis type IVB (MPS IVB); acid β-galactosidasedeficiency); Pompe disease (acid α-glucosidase deficiency); Sandhoffdisease (β-hexosaminidase B deficiency); Sanfilippo syndrome type A(mucopolysaccharidosis type IIIA (MPS IIIA); heparan-N-sulfatasedeficiency); Sanfilippo syndrome type B (mucopolysaccharidosis type IIIB(MPS IIIB); alpha-N-acetylglucosaminidase deficiency); Sanfilipposyndrome type C (mucopolysaccharidosis type IIIC (MPS IIIC);acetyl-CoA:α-glucosaminide N-acetyltransferase deficiency); Sanfilipposyndrome type D (mucopolysaccharidosis type IIID (MPS IIID);N-acetylglucosamine-6-sulfate sulfatase deficiency); Schindler/Kanzakidisease (alpha-N-acetylgalactosaminidase deficiency); sialidosis(sialidase deficiency); Sly syndrome (mucopolysaccharidosis type VII(MPS VII); β-glucuronidase deficiency); and Tay-Sachs disease(β-hexosaminidase A deficiency).

In particular variations, a composition comprising (i) a polymer ofFormula I wherein G is present and is a cationic peptide, polyamine, orpolycation (e.g., a copolymer of formula VII) and (ii) an mRNA encodingan ornithine transcarbamylase (OTC) protein is used in a method to treatornithine transcarbamylase deficiency (OTCD). OTCD is a urea cycledisorder that can trigger hyperammonemia, a life-threatening illnessthat leads to brain damage, coma or even death. This is due todeficiency in the activity of OTC, a key enzyme in the urea cycle, whichprimarily takes place in the liver and is responsible for removal ofexcess nitrogen in the body. Ammonium nitrogen is produced from proteinintake as well as protein breakdown in the body. In the liver, thisammonium nitrogen is converted into urea by enzymes in the urea cycle.Urea is non-toxic and cleared easily through the kidneys in urine,normally. However when the OTC enzyme is deficient, ammonia levels risein blood and cause severe brain damage. Patients with severe OTCdeficiency are most often identified 2-3 days after birth where thepatient has significantly elevated blood ammonia levels and ends up in acoma. Patients with milder OTC deficiency can have crises during timesof stress resulting in elevated ammonia levels that can also lead tocoma. Current therapies include ammonia scavenger drugs (Buphenyl,Ravicti) for use in patients with hyperammonemia.

The OTC gene is X-linked. The disease is present in males with onemutant allele and in females either homozygous or heterozygous withmutant alleles. Male patients are typically those with the severest OTCdeficiency found right after birth. In addition to elevation in bloodammonia levels, urinary orotic acid levels are also elevated. Inpatients with severe OTC deficiency, OTC enzyme activity is <20% ofnormal levels. In patients with milder OTC deficiency, OTC enzymeactivity is up to 30% of normal levels.

A method for treating OTCD with a composition comprising an OTC-encodingmRNA and a copolymer of the present disclosure generally includesadministering to a subject having OTCD a therapeutically effectiveamount of the composition, whereby the OTC-encoding mRNA is delivered toliver cells and translated during protein synthesis to produce the OTCprotein. The OTC-encoding mRNA may be an mRNA as set forth above withrespect to a composition or method for increasing OTC protein in a cell.In particular variations, the copolymer is a copolymer as set forth inany one of (a)-(z) above with respect to a composition or method forincreasing OTC protein in a cell.

The efficacy of a copolymer/mRNA composition for treating a disease canbe evaluated in vivo in animal models of disease. Particularly suitableanimal models for evaluating efficacy of a copolymer/mRNA compositionfor treatment of OTCD includes known mouse models having deficiencies ofthe OTC enzyme in the liver. One such mouse model, OTC^(spf-ash) (sparsefur and abnormal skin and hair) mice, contain an R129H mutationresulting in reduced levels of OTC protein and have only 5-10% of thenormal level of enzyme activity in liver (see Hodges et al., PNAS86:4142-4146, 1989). Another model, OTC^(spf) mice, contain an H117Nmutation which results in reduced levels of enzyme activity to 5-10% ofnormal levels (see Rosenberg et al., Science 222:426-428, 1983). Both ofthese mouse models have elevated urine orotic acid levels compared totheir wild-type littermate mice. A third model for OTC deficiency isinducing hyperammonemia in OTC^(spf) or OTC^(spf-ash) mice (Cunninghamet al., Mol Ther 19(5): 854-859, 2011). These mice are treated with OTCsiRNA or AAV2/8 vector/OTC shRNA to knockdown residual endogenous OTCexpression and activity. Plasma ammonia levels are elevated and mice dieapproximately 2-14 days.

In additional variations, a composition comprising (i) a polymer ofFormula I wherein G is present and is a cationic peptide, polyamine, orpolycation (e.g., a copolymer of formula VII) and (ii) an mRNA encodingan enzyme deficient in an organic acidemia, or a subunit of the enzyme,is used to treat the organic acidemia. Organic acidemia (also known asaciduria) (OA) is a group of disorders characterized by the excretion ofnon-amino organic acids in the urine. Most organic acidemias result fromdysfunction of a specific step in amino acid catabolism, usually theresult of deficient enzyme activity. The majority of organic aciddisorders are caused by abnormal amino acid catabolism of branched-chainamino acids or lysine. They include propionic acidemia (PA),methylmalonic acidemia (MMA), maple syrup urine disease (MSUD), andothers. These organic acidemias are inherited in an autosomal recessivemanner. A neonate affected with an OA is usually well at birth and forthe first few days of life. The usual clinical presentation is that oftoxic encephalopathy and includes vomiting, poor feeding, neurologicsymptoms such as seizures and abnormal tone, and lethargy progressing tocoma. Outcome can be improved by diagnosis and treatment in the firstten days of life. In the older child or adolescent, variant forms of theOAs can present as loss of intellectual function, ataxia or other focalneurologic signs, Reye syndrome, recurrent ketoacidosis, or psychiatricsymptoms.

Clinical laboratory findings indicate that organic acidemias includeacidosis, ketosis, hyperammonemia, abnormal liver function,hypoglycemia, and neutropenia. First-line diagnosis in the organicacidemias is urine organic acid analysis using gas chromatography withmass spectrometry (GC/MS). The urinary organic acid profile is nearlyalways abnormal in the face of acute illness. Confirmatory testinginvolves assay of the activity of the deficient enzyme in lymphocytes orcultured fibroblasts and/or molecular genetic testing. Characteristicsof the three primary disorders are summarized in Table 5.

TABLE 5 Metabolic Findings in Organic Acidemias Caused by Abnormal AminoAcid Catabolism Diagnostic Analytes Amino Acid by GC/MS and Pathway(s)Quantitative Amino Disorder Affected Enzyme Acid Analysis Propionicacidemia Isoleucine, valine, Propionyl CoA Propionic acid, 3-OH (PA)methionine, carboxylase (PCC) propionic acid, threonine (composed ofthree methyl citric acid, PCCA subunits and propionyl glycine in threePCCB urine subunits) Propionyl carnitine, increased glycine in bloodMethylmalonic Isoleucine, valine, Methylmalonyl CoA Methylmalonic acidacidemia (MMA) methionine, mutase (MUT) in blood and urine threoninePropionic acid, 3-OH propionic acid, methyl citrate in urine Acylcarnitines, increased glycine in blood Maple syrup urine Leucine,isoleucine, Branched-chain Branched-chain disease (MSUD) valine ketoacidketoacids and dehydrogenase hydroxyacids in urine (BCKDH) Alloisoleucinein (composed of four plasma different subunits)

Once the detection of specific analytes narrows the diagnosticpossibilities, the activity of the deficient enzyme is assayed inlymphocytes or cultured fibroblasts as a confirmatory test. For manypathways, no single enzyme assay can establish the diagnosis. Forothers, tests such as complementation studies need to be done.

The goal of therapy is to restore biochemical and physiologichomeostasis. Neonates require emergency diagnosis and treatmentdepending on the specific biochemical lesion, the position of themetabolic block, and the effects of the toxic compounds. Treatmentstrategies include: (1) dietary restriction of the precursor amino acidsand (2) use of adjunctive compounds to (a) dispose of toxic metabolitesor (b) increase activity of deficient enzymes. Liver transplantation hasbeen successful in a small number of affected individuals. Even withcurrent clinical management approaches, individuals with organicacidemias have a greater risk of infection and a higher incidence ofpancreatitis, which can be fatal.

Enzyme replacement therapy via specific mRNA delivery to the liveroffers the most effective treatment of the organic acidemias. In certainembodiments of a method for treating an organic acidemia, a compositioncomprising (i) a polymer of Formula I wherein G is present and iscationic peptide and (ii) an mRNA encoding a methylmalonyl CoA mutase(MUT) is used to treat methylmalonic acidemia MMA. In other embodiments,a composition comprising (i) a polymer of Formula I wherein G is presentand is cationic peptide and (ii) an mRNA encoding a PCC subunit (PCCA orPCCB) is used to treat propionic acidemia (PA). In yet otherembodiments, a composition comprising (i) a polymer of Formula I whereinG is present and is cationic peptide and (ii) an mRNA encoding a BCKDHsubunit is used to treat maple syrup urine disease (MSUD). A method fortreating MMA, PA, or MSUD with a composition comprising an Mut, Pcca/b,or BCKDH subunit mRNA and a copolymer of the present disclosuregenerally includes administering to a subject having an organic acidemiaof the specified type a therapeutically effective amount of thecomposition, whereby the Mut, Pcca/b, or BCKDH subunit mRNA is deliveredto liver cells and translated during protein synthesis to produce therespective protein. A Mut or Pcca/b mRNA may be an mRNA as set forthabove with respect to a composition or method for increasing therespective protein in a cell. In particular variations, the copolymer isa copolymer as set forth in any one of (a)-(z) above with respect to acomposition or method for increasing MUT, PCC, or BCKDH protein.

The efficacy of a copolymer/mRNA composition for treating an organicacidemia disease can be evaluated in vivo in animal models of disease.For example, particularly suitable animal models for evaluating efficacyof a copolymer/mRNA composition for treatment of MMA and PA are asfollows. Mut^(−/−) neonatal mice with a severe form of MMA, whichnormally die within the first 21 days of life, have been successfullytreated with hepatocyte-directed delivery of the methylmalonyl-CoAmutase (Mut) gene. Following an intrahepatic injection ofadeno-associated virus expressing the murine Mut gene, Mut^(−/−) micewere rescued and lived beyond 1 year of age (Carrillo-Carrasco et al.,Hum. Gene Ther. 21:1147-1154, 2010). Another MMA disease model wheremice survive into adulthood is Mut^(−/−) mice with Mut cDNA expressedunder the control of an insulated, muscle-specific promoter; Mut^(−/−);Tg^(INS-MCK-Mut) (Manoli et al., 2011, SIMD Abstract). These mice haveelevated plasma methylmalonic acid levels and decreased oxidativecapacity as measured by a ¹³C propionate oxidation/breathe assay. Amouse model of PA (Pcca^(−/−) mice) succumbs to death 24-36 h afterbirth and is associated with fatal ketoacidosis (Miyazaki et al., J.Biol. Chem. 276:35995-35999, 2001). Pcca gene transfer that provides apostnatal PCC activity of 10-20% in the liver of a transgenic mousestrain attenuates the fatal ketoacidosis in newborn mice (Miyazaki etal., 2001, supra). Recently, an intrahepatic adeno-associated virusmediated gene transfer for human Pcca was tested in neonatal Pcca^(−/−)mice (Chandler et al., Hum. Gene Ther. 22:477-481, 2010). The authorsfound a sustained therapeutic effect as demonstrated in a survival rateof approximately 64% and reduction of disease-related metabolites(Chandler et al., 2010, supra). Another mouse disease model of PA is ahypomorphic model where Pcca^(−/−) mice express a transgene bearing anA138T mutant of the PCCA protein. These mice have 2% of wild-type PCCactivity, survive to adulthood and have elevations in disease-relatedmetabolites (Guenzel et al., Mol. Ther. 21:1316-1323, 2013). Treatmentof these mice with adeno-virus or AAV vector expressing human PCCA cDNAresulted in increased PCC enzyme activity and correction of diseasemarker levels (Guenzel et al., 2013, supra). Taken together, in murinemodels of MMA and PA gene transfer approaches rescue neonatal mice orrestore enzyme activity and correct disease metabolite levels in adultdisease models thereby permitting evaluation of mRNA delivery forrestoration of the defective enzymes.

In certain embodiments, copolymers of the present invention are alsouseful in the preparation of a medicament for the treatment of a diseaseor condition associated with defective gene expression and/or activityin a subject.

In certain embodiments, copolymers of the present invention are alsouseful in the preparation of a medicament for the treatment of a diseaseor condition associated with deficiency in a functional polypeptide.

In any of the above described methods of treating a disease or conditionassociated with defective gene expression and/or activity, the gene is,but is not limited to, a growth factor or growth factor receptor gene,an gene encoding an enzyme (for example, a phosphatase or a kinase,e.g., a protein tyrosine, serine, or threonine kinase), an adaptorprotein gene, a gene encoding a G protein superfamily molecule, or agene encoding a transcription factor.

Examples of suitable gene targets useful in the methods of treating adisease or condition associated with defective gene expression and/oractivity as described herein include the following genes or genesencoding the following proteins MEX3, MMP2, ApoB, ERBB2, VascularEndothelial Growth Factor (VEGF), Vascular Endothelial Growth FactorReceptor (VEGFR), Platelet Derived Growth Factor Receptor (PDGF), ABL,KITT, FMS-like tyrosine kinase 3 (FLT3), Cav-1, Epidermal Growth FactorReceptor (EGFR), H-Ras, K-Ras, N-Ras, Bc1-2, Survivin, FAK, STAT-3,HER-3, Beta-Catenin, ornithine transcarbamylase, alpha-1-antitrypsin,and Src.

Other examples of suitable gene targets useful in the methods oftreating a disease or condition associated with defective geneexpression and/or activity as described herein include tumorsuppressors, where loss of function of the mutated gene can be correctedby delivery of mRNA encoding the functional protein to treat cancer.Suitable tumor suppressor targets include Retinoblastoma protein (pRb),p53 tumor-suppressor protein, Phosphatase and tensin homolog (PTEN), VonHippel-Lindau tumor suppressor (pVHL), Adenomatous polyposis coli (APC),FAS receptor (FasR), Suppression of tumorigenicity 5 (ST5), YPEL3,Suppressor of tumorigenicity protein 7 (ST7), and Suppressor oftumorigenicity 14 protein (ST14).

Copolymers as described herein can be formulated into pharmaceuticalcompositions. In certain embodiments, the present invention provides forpharmaceutical compositions which comprise, as active ingredient, ablock copolymer of Formula I, Formula III, Formula IV, Formula VI, orFormula VII. Typically, pharmaceutical compositions of the presentinvention include a block copolymer of Formula I, Formula III, FormulaIV, Formula VI, or Formula VII and pharmaceutically acceptable carriers,diluents and/or excipients.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The block copolymers of Formula I and pharmaceutical compositionsprepared from them can be administered in a wide variety of routes ofadministration such as parenteral, oral, topical, rectal, inhalation andthe like. Formulations will vary according to the route ofadministration selected. Examples are oral and parenteral dosage forms.Thus, the block copolymers of Formula I and pharmaceutical compositionsprepared from them can be administered by injection, that is,intravenously, intramuscularly, intracutaneously, subcutaneously,intraduodenally, or intraperitoneally. Also, the block copolymers ofFormula I and pharmaceutical compositions prepared from them can beadministered by inhalation, for example, intranasally. Additionally, thecompounds of the present disclosure can be administered transdermally.The following dosage forms may comprise as the active component, a blockcopolymer of Formula I or as active component complexed to it such as anoligonucleotide—for example mRNA.

For preparing pharmaceutical compositions from block copolymers ofFormula I, pharmaceutically acceptable carriers can be either solid orliquid. Solid form preparations include powders, tablets, pills,capsules, cachets, suppositories, and dispersible granules. A solidcarrier can be one or more substances which may also act as diluents,flavoring agents, binders, preservatives, tablet disintegrating agents,or an encapsulating material.

The pharmaceutical composition is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The quantity of polymer in a unit dose preparation may be varied oradjusted, for example from about 0.1 mg/kg to about 200 mg/kg,preferably from about 0.5 mg/kg to about 100 mg/kg, with the associatedoligonucleotide (e.g., mRNA) varied or adjusted from about 0.001 mg/kgto about 10 mg/kg, preferably from about 0.1 mg/kg to about 5 mg/kg,according to the particular application and the potency of the activecomponent.

The pharmaceutical compositions disclosed herein can, if desired, alsocontain other compatible therapeutic agents. For example, copolymers asdescribed herein can be formulated into pharmaceutical compositions thatinclude a second active ingredient such as a chemotherapeutic agent.Chemotherapeutic agents can also be coadministered with the presentlydescribed copolymers. Such coadministration could include sequentialadministration.

In therapeutic use as agents for the treatment of disease, the polymersutilized in the pharmaceutical methods of this invention can beadministered at an initial dosage of about 0.01 mg to about 200 mg/kgdaily, with the associated oligonucleotide (e.g., mRNA) at an initialdose of about 0.001 mg to about 10 mg/kg daily. A daily polymer doserange of about 0.01 mg to about 100 mg/kg, about 0.1 mg to about 100mg/kg, or about 0.1 mg to about 50 mg/kg is preferred. Anoligonucleotide (e.g. mRNA), formulated with the polymer, may beadministered at a daily dose of, for example, about 0.001 mg to about 5mg/kg, about 0.01 mg to about 5 mg/kg, about 0.1 mg to about 5 mg/kg,about 0.01 mg to about 10 mg/kg, or about 0.1 mg to about 10 mg/kg. Thedosages, however, may be varied depending upon the requirements of thepatient, the severity of the condition being treated, and the compoundbeing employed.

Determination of the proper dosage for a particular situation is withinthe skill of the art. Determination of a therapeutically effectivedosage is typically based on animal model studies followed up by humanclinical trials and is guided by determining effective dosages andadministration protocols that significantly reduce the occurrence orseverity of the subject disease or condition in model subjects.Effective doses of the compositions of the present disclosure varydepending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, aswell as the specific activity of the composition itself and its abilityto elicit the desired response in the individual. Usually, the patientis a human, but in some diseases, the patient can be a nonhuman mammal.Typically, dosage regimens are adjusted to provide an optimumtherapeutic response, i.e., to optimize safety and efficacy.Accordingly, a therapeutically effective amount is also one in which anyundesired collateral effects are outweighed by beneficial effects ofadministering a composition. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under the circumstances is reached. Effective dosages canbe achieved by single or multiple administrations, including, e.g.,multiple administrations per day or daily, weekly, bi-weekly, or monthlyadminitrations. For example, a total daily dosage may be divided andadministered in portions during the day, if desired. In certainvariations, a regimen consists of an initial administration followed bymultiple, subsequent administrations at semi-weekly, weekly, orbi-weekly intervals. Another regimen consists of an initialadministration followed by multiple, subsequent administrations atmonthly or bi-monthly intervals. Alternatively, administrations can beon an irregular basis as indicated by monitoring of clinical symptomsand/or physiological correlates of the disease or condition.

Examples of pharmaceutical compositions of the present invention includethose comprising a block copolymer of Formula I and a pharmaceuticallyacceptable diluent or carrier, wherein Q is S-S-oligonucleotide,

and

designates a point of attachment.

Another example of a pharmaceutical composition of the present inventionincludes a pharmaceutical composition comprising (a) a block copolymerof Formula I wherein G is present and is a cationic peptide, polyamine,or polycation, (b) an mRNA molecule and (c) a pharmaceuticallyacceptable diluent or carrier. In some such embodiments, the blockcopolymer of Formula I is a copolymer of formula VII.

Additional examples of pharmaceutical compositions of the presentinvention includes pharmaceutical compositions comprising (a) a blockcopolymer of Formula I wherein G is present and is a cationic peptide,polyamine, or polycation, (b) an mRNA molecule and (c) apharmaceutically acceptable diluent or carrier, where the mRNA moleculeis complexed to the cationic peptide, polyamine, or polycation. In somesuch embodiments, the block copolymer of Formula I is a copolymer offormula VII.

Additional examples of pharmaceutical compositions of the presentinvention includes pharmaceutical compositions comprising (a) a blockcopolymer of Formula I wherein G is present and is cationic peptide, (b)an mRNA molecule and (c) a pharmaceutically acceptable diluent orcarrier, where the mRNA molecule is complexed to the cationic peptideand the nitrogen to phosphorous ratio between the cationic peptide andmRNA is between 100:1 and 1:1. Other examples include pharmaceuticalcompositions where the nitrogen to phosphorous ratio between thecationic peptide and mRNA is between 50:1 and 1:1. Other examplesinclude pharmaceutical compositions where the nitrogen to phosphorousratio between the cationic peptide and mRNA is between 20:1 and 1:1.Other examples include pharmaceutical compositions where the nitrogen tophosphorous ratio between the cationic peptide and mRNA is between 30:1and 10:1. Other examples include pharmaceutical compositions where thenitrogen to phosphorous ratio between the cationic peptide and mRNA isbetween 25:1 and 15:1. In some such embodiments as above, the blockcopolymer of Formula I is a copolymer of formula VII.

Polymers described here are prepared in any suitable manner. Suitablesynthetic methods used to produce the polymers provided herein include,by way of non-limiting example, cationic, anionic and free radicalpolymerization. In some instances, when a cationic process is used, themonomer is treated with a catalyst to initiate the polymerization.Optionally, one or more monomers are used to form a copolymer. In someembodiments, such a catalyst is an initiator, including, e.g., protonicacids (Bronsted acid) or Lewis acids, in the case of using Lewis acidsome promoter such as water or alcohols are also optionally used. Insome embodiments, the catalyst is, by way of non-limiting example,hydrogen iodide, perchloric acid, sulfuric acid, phosphoric acid,hydrogen fluoride, chlorosulfonic acid, methansulfonic acid,trifluoromehtanesulfonic acid, aluminum trichloride, alkyl aluminumchlorides, boron trifluoride complexes, tin tetrachloride, antimonypentachloride, zinc chloride, titanium tetrachloride, phosphorouspentachloride, phosphorus oxychloride, or chromium oxychloride. Incertain embodiments, polymer synthesis is performed neat or in anysuitable solvent. Suitable solvents include, but are not limited to,pentane, hexane, dichloromethane, chloroform, or dimethyl formamide(DMF). In certain embodiments, the polymer synthesis is performed at anysuitable reaction temperature, including, e.g., from about −50° C. toabout 100° C., or from about 0° C. to about 70° C.

In some embodiments, polymerization processes are carried out in aliving mode, in any suitable manner, such as but not limited to AtomTransfer Radical Polymerization (ATRP), nitroxide-mediated living freeradical polymerization (NMP), ring-opening polymerization (ROP),degenerative transfer (DT), or Reversible Addition FragmentationTransfer (RAFT). Using conventional and/or living/controlledpolymerizations methods, various polymer architectures can be produced,such as but not limited to block, graft, star and gradient copolymers,whereby the monomer units are either distributed statistically or in agradient fashion across the chain or homopolymerized in block sequenceor pendant grafts. In other embodiments, polymers are synthesized byMacromolecular design via reversible addition-fragmentation chaintransfer of Xanthates (MADIX) (“Direct Synthesis of Double HydrophilicStatistical Di- and Triblock Copolymers Comprised of Acrylamide andAcrylic Acid Units via the MADIX Process”, Daniel Taton, et al.,Macromolecular Rapid Communications, 22, No. 18, 1497-1503 (2001)).

In certain embodiments, Reversible Addition-Fragmentation chain Transferor RAFT is used in synthesizing ethylenic backbone polymers of thisinvention. RAFT is a living polymerization process. RAFT comprises afree radical degenerative chain transfer process. In some embodiments,RAFT procedures for preparing a polymer described herein employsthiocarbonylthio compounds such as, without limitation, dithioesters,dithiocarbamates, trithiocarbonates and xanthates to mediatepolymerization by a reversible chain transfer mechanism. In certaininstances, reaction of a polymeric radical with the C═S group of any ofthe preceding compounds leads to the formation of stabilized radicalintermediates. Typically, these stabilized radical intermediates do notundergo the termination reactions typical of standard radicalpolymerization but, rather, reintroduce a radical capable ofre-initiation or propagation with monomer, reforming the C═S bond in theprocess. In most instances, this cycle of addition to the C═S bondfollowed by fragmentation of the ensuing radical continues until allmonomer has been consumed or the reaction is quenched. Generally, thelow concentration of active radicals at any particular time limitsnormal termination reactions.

In some embodiments, polymers of the present invention have a lowpolydispersity index (PDI) or differences in chain length.Polydispersity index (PDI) can be determined in any suitable manner,e.g., by dividing the weight average molecular weight of the polymerchains by their number average molecular weight. The number averagemolecule weight is sum of individual chain molecular weights divided bythe number of chains. The weight average molecular weight isproportional to the square of the molecular weight divided by the numberof molecules of that molecular weight. Since the weight averagemolecular weight is always greater than the number average molecularweight, polydispersity is always greater than or equal to one. As thenumbers come closer and closer to being the same, i.e., as thepolydispersity approaches a value of one, the polymer becomes closer tobeing monodisperse in which every chain has exactly the same number ofconstitutional units. Polydispersity values approaching one areachievable using radical living polymerization. Methods of determiningpolydispersity, such as, but not limited to, size exclusionchromatography, dynamic light scattering, matrix-assisted laserdesorption/ionization chromatography and electrospray masschromatography are well known in the art. In some embodiments, thepolymers (e.g., membrane destabilizing polymers) provided herein have apolydispersity index (PDI) of less than 2.0, or less than 1.8, or lessthan 1.6, or less than 1.5, or less than 1.4, or less than 1.3, or lessthan 1.2. In some embodiments, the polymer is a block copolymer (e.g.,membrane destabilizing block copolymers) comprising a hydrophilic blockand a hydrophobic block and having a polydispersity index (PDI) of lessthan 2.0, or less than 1.8, or less than 1.6, or less than 1.5, or lessthan 1.4, or less than 1.3, or less than 1.2.

Polymerization processes described herein optionally occur in anysuitable solvent or mixture thereof. Suitable solvents include water,alcohol (e.g., methanol, ethanol, n-propanol, isopropanol, butanol),tetrahydrofuran (THF) dimethyl sulfoxide (DMSO), dimethylformamide(DMF), acetone, acetonitrile, hexamethylphosphoramide, acetic acid,formic acid, hexane, cyclohexane, benzene, toluene, dioxane, methylenechloride, ether (e.g., diethyl ether), chloroform, and ethyl acetate. Inone aspect, the solvent includes water, and mixtures of water andwater-miscible organic solvents such as DMF.

The copolymers of this invention can be made by processes which includeprocesses analogous to those known in the chemical arts, particularly inlight of the description contained herein. Certain processes for themanufacture of the copolymers of this invention are provided as furtherfeatures of the invention and are illustrated by the following examplesand as described in the experimental section.

An example of a process for the preparation of a block copolymer ofFormula I includes

-   a) contacting a compound of Structure Va, Vb, Vc, or Vd

where R²⁷=C₁-C₁₂ alkyl,

where R²⁸=C₁-C₁₂ alkyl,

where R²⁵ and R²⁶ are independently H, alkyl, aryl, or heteroaryl,

where T1 is absent or a first targeting moiety and L1 is absent or alinking moiety; with one or more monomers selected from monomers of theformulae A1, A2 and A3

where R¹ is H or C₁-C₆ alkyl, R² is O, NH or N(C₁-C₆ alkyl), Q is —SR²⁰or S-S-pyridyl, and R²⁰ is a thiol-protecting group;

where n is 1-120, R³ is H or C₁-C₆ alkyl, R⁴ is S, O, NH or N(C₁-C₆alkyl), R⁵ is O or S and R⁶ is H or C₁-C₆ alkyl;

where R⁷ and R¹⁰ are independently H or C₁-C₆ alkyl, R⁸ is S, O, NH orN(C₁-C₆ alkyl), and R⁹ is O or S and R^(H) is an amine protecting group;in the presence of a free radical;

-   b) contacting the product of step a) with monomers of formulae B1,    B2 and B3

where R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H or C₁-C₆ alkyl; inthe presence of a free radical; and

-   c) deprotecting the product of step b) and contacting it with an    oligonucleotide, cationic peptide, polyamine, or polycation    comprising a thiol-reactive or amine-reactive group; or contacting    the product of step b) with an oligonucleotide, cationic peptide,    polyamine, or polycation comprising a thiol group. In some    embodiments of a process as above, for the monomer of formula A2, n    is 1-20.

In one example the synthetic process described above is carried outwhere compound Va is

the monomer of formula A1 is

the monomer of formula A2 is

the monomer of formula A3 is

wherein R¹¹ is an amine protecting group; the monomer of formula B1 isbutyl methacrylate; the monomer of formula B2 is 2-propyl acrylic acid;and the monomer of formula B3 is 2-(dimethylamino)ethyl methacrylate.

Another example of a process for the preparation of a block copolymer ofFormula I includes

-   a) contacting a compound of Structure Va, Vb, Vc, or Vd,

where R²⁷=C₁-C₁₂ alkyl,

where R²⁸=C₁-C₁₂ alkyl,

where R²⁵ and R²⁶ are independently H, alkyl, aryl, or heteroaryl,

where T1 is absent or a first targeting moiety and L1 is absent or alinking moiety; with one or more monomers selected from monomers of theformulae A2, A4 and A5,

where n is 1-120, R³ is H or C₁-C₆ alkyl, R⁴ is S, O, NH or N(C₁-C₆alkyl), R⁵ is O or S and R⁶ is H or C₁-C₆ alkyl;

where R¹⁷ is H or C₁-C₆ alkyl, R¹⁸ is O, S, NH or N(C₁-C₆ alkyl), R¹⁹ isO or N, R²⁰ is H, T2, or C₁-C₆ alkyl, where T2 is a second targetingmoiety;

where R²¹ is H or C₁-C₆ alkyl, R²² is O, NH or N(C₁-C₆ alkyl), R²³ is H,aryl, arylhalide, alkyl, alkyl alcohol; in the presence of a freeradical;

-   b) contacting the product of step a) with monomers of formulae B1,    B2, B3, and B4,

where R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are independently H or C₁-C₆alkyl, R¹⁸ is O, S, NH or N(C₁-C₆ alkyl), and Q is —SR²⁰ or S-S-pyridyl,and R²⁰ is a thiol-protecting group; in the presence of a free radical;and

-   c) deprotecting the product of step b) and contacting it with an    oligonucleotide, cationic peptide, polyamine, or polycation    comprising a thiol-reactive or amine-reactive group; or contacting    the product of step b) with an oligonucleotide, cationic peptide,    polyamine, or polycation comprising a thiol group. In some    embodiments of a process as above, for the monomer of formula A2, n    is 1-20.

In one example the synthetic process described above is carried outwhere the monomer of formula A2 is

the monomer of formula B1 is butyl methacrylate; the monomer of formulaB2 is 2-propyl acrylic acid; the monomer of formula B3 is2-(dimethylamino)ethyl methacrylate; and the monomer of formula B4 is

In some embodiments of a process as above for the preparation of a blockcopolymer of Formula I where the product of step b) is contacted with acationic peptide, polyamine, or polycation comprising a thiol-reactiveor amine-reactive group, or with a cationic peptide, polyamine, orpolycation comprising a thiol group, the process further includescontacting the product of step c) with a polynucleotide (e.g., an mRNA)to form a complex comprising the block copolymer of Formula I and thepolynucleotide. In particular variations of a method as above, R²⁵and/or R²⁶ of Structure Vc is a heteroaryl having the structure

Tri-NAG structures can be constructed in a variety of ways. One suchmethod starts withdi-tert-butyl-4-amino-4[2-(tertbutoxycarbonyl0ethyl]heptanedionate. Fmocprotection of the tertiary amine, followed by removal of the t-butylesters results in a branched tri-carboxylic acid. The tri-carboxylicacid can be activated for an amidation reaction with trifluoroaceticacid pentafluorophenyl ester. The resulting tri-pentaluorophenyl estercan be reacted with (2-amino-ethocy)-acetic acid to afford a chainextended tri-carboxylic acid. The tri-carboxylic acid can again beactivated for an amidation reaction with trifluoroacetic acidpentafluorophenyl ester. The resulting tri-pentaluorophenyl ester can bereacted with NAc-Galactosamine-C₅—NH₂ (or the O-Ac protected sugar), toafford the tri-NAG derivative as the Fmoc protected amine. The resultingFmoc protected amine can be deprotected to the teritiary amine. At thisstage, the tri-NAG amine can be coupled to HO₂C-PEGx-ECT to make atri-NAG-PEGx-ECT chain transfer agent in a similar manor to other chaintransfer agents described herein. Alternatively, the tri-NAG amine canbe acylated with a PEGx amino acid, wherein the amino functionality isprotected, for example as the TFA amide. Following deprotection of theamine, the amine can be amidated with 4-formylbenzoic acid that isactivated for the amidation reaction (for example as the NHS ester). Theresulting tri-NAG-PEGx-Ph-aldehyde can then be added to a chain transferagent or a polymer that has a hydroxylamine group to form an oxime. Itis understood in the above reaction sequence that PEGx is meant to bePEG where x=2-460.

EXAMPLES

Throughout this description, various known acronyms and abbreviationsare used to describe monomers or monomeric residues derived frompolymerization of such monomers. Without limitation, unless otherwisenoted: “BMA” (or the letter “B” as equivalent shorthand notation)represents butyl methacrylate or monomeric residue derived therefrom;“DMAEMA” (or the letter “D” as equivalent shorthand notation) representsN,N-dimethylaminoethyl methacrylate or monomeric residue derivedtherefrom; “PAA” (or the letter “P” as equivalent shorthand notation)represents 2-propylacrylic acid or monomeric residue derived therefrom;“PEGMA_(n)”, wherein n=8-9 or 4-5, refers to the pegylated methacrylicmonomer, CH₃O(CH₂CH₂O)_(n)C(O)C(CH₃)CH₂ or monomeric residue derivedtherefrom; “PDSMA” represents 2-(pyridin-2-yldisulfanyl)ethylmethacrylate or monomeric residue derived therefrom; “TFPMA” represents2,3,5,6-tetrafluorphenyl methacrylate or monomeric residue derivedtherefrom; “PFPMA” represents pentafluorophenyl methacrylate ormonomeric residue derived therefrom. In each case, any such designationindicates the monomer (including all salts, or ionic analogs thereof),or a monomeric residue derived from polymerization of the monomer(including all salts or ionic analogs thereof), and the specificindicated form is evident by context to a person of skill in the art.Figures of polymers or macro CTAs in the following examples are notmeant to describe any particular arrangement of the constitutional unitswithin a particular block. “KDa” and “k” as used herein refer tomolecular weight in kilodaltons.

Structures of the monomers used in the preparation of the polymers:

¹H NMR spectra of the monomers and polymers were recorded on BrukerAV301 or Varian 400 MHz in deuterated solvents as indicated in eachexperiment at 25° C. Mass spectra was acquired on Bruker Esquire IonTrap instrument using the following settings: electro-spray ionization,capillary exit voltage of 100.0 V, scanning from 80.00 m/z to 2200.00m/z, dry gas flow of 6.0 L/min. Mass spectroscopy was also conducted onan 6520 Accurate Mass Q-TOF LC/MS equipped with an Agilent 1290 InfinityUHPLC system with UV detector. Gel permeation chromatography (GPC) wasused to determine molecular weights and polydispersities (PDI,M_(w)/M_(n)) of the copolymer samples in DMF using a Viscotek GPCmaxVE2001 and refractometer VE3580 (Viscotek, Houston, Tex.). HPLC-gradedimethylformamide (DMF) containing 1.0 wt % LiBr was used as the mobilephase. UV/Vis spectroscopy was performed using a NanoDrop UV/Visspectrometer (path length 0.1 cm). Particle sizes of the polymers andpolymer-siRNA conjugate particles were measured by dynamic lightscattering using a Malvern Zetasizer Nano ZS. HPLC analysis wasperformed on Shimadzu LD-20AB with the variable-wavelength UV detectorwith a C18 analytical reverse phase column (ES Industries ChromegaColumns, Sonoma C18 catalog number 155B21-SMA-C18(2), 100 Å, 25.0 cm×4.6mm, column heated to 30° C.). All reagents were from commercial sources,unless indicated otherwise, and the monomers were purified from tracesof stabilizing agents prior to use in the polymerization reactions.Cyano-4-(ethylsulfanylthiocarbonyl) sulfanylpentanoic acid (ECT) wasobtained from Omm Scientific. Azobisisobutyronitrile (AIBN) (Wakochemicals) was used as the radical initiator in all polymerizationreactions, unless stated otherwise.

Example 1. Synthesis of PEG_(0.6k)-CTA (Compound 6)

HOOC-PEG_(0.6K)-ECT (Compound 6). To a 100 mL one-neck round-bottomflask was added ECT (473 mg, 2.0 mmol, Omm Scientific) followed byanhydrous tetrahydrofuran (20 mL) and triethylamine (0.307 mL, 2.2mmol). This mixture was stirred at 0° C. for 5 min beforetrifluoroacetic acid pentafluorophenyl ester (0.368 mL, 2.14 mmol) wasadded drop wise to the stirred reaction. The mixture was stirred at 0°C. for 5 min then warmed to room temperature.

After allowing to react for 20 min at room temperature, the reaction wasdiluted into EtOAc (100 mL) and extracted with saturated aqueoussolution of NaHCO₃ (3×40 mL). The EtOAc layer was separated, dried overNa₂SO₄, filtered and then evaporated providing the crude PFP-ester 4 asyellow oil.

The crude ester 4 was dissolved in anhydrous CH₂Cl₂ (20 mL) and thencooled to 0° C. To the cooled stirred solution was added triethylamine(0.251 mL, 1.8 mmol) and Amino-dPEG12-acid (1.12 g, 1.8 mmol, QuantaBiodesign), and the mixture was warmed to room temperature. Afterstirring for 20 min at room temperature, the reaction mixture wasevaporated using a rotary evaporator providing yellow oil. The yellowoil was dissolved in CH₂Cl₂ (approximately 2 mL) and the product waspurified by flash chromatography (SiO₂, column size 5.0 cm ID×10.0 cmlength; isocratic elution with 100% CH₂Cl₂ for 500 mL; then CH₂Cl₂/MeOH,20:1 v/v for 500 mL; then CH₂Cl₂/MeOH, 10:1 v/v for 3.0 L). Theproduct-containing fractions, as determined by TLC, were combined, andthe solvent was removed by rotary evaporation providing 750 mg (48%) ofthe desired compound 6 as orange oil. ¹H NMR (CD3OD): δ 1.35 (t, 3H,J=7.5 Hz, CH₃), 1.89 (s, 3H, CH₃), 2.38-2.57 (m, 6H), 3.32-3.41 (m, 4H),3.50-3.75 (m, 48H).

Example 2. Synthesis of Nag(OAc4)C5N-PEG-_(0.6K)-CTA (Compound 8)

Step 1. Synthesis of Compound 3.

N-t-Boc-5-amino-1-pentanol. To a 1.0 L one-neck round-bottom flaskcontaining a solution of 5-amino-1-pentanol (15.0 g, 145.4 mmol) inwater (140 mL) and saturated aqueous NaHCO₃ (1.4 mL), a solution ofdi-tert-butyl dicarbonate (33.3 g, 152.7 mmol) in THF (280 mL) wasadded. The mixture was then stirred at room temperature overnight withthe flask open to the atmosphere. The reaction mixture was diluted withsaturated aqueous NaHCO₃ (90 mL) and extracted with EtOAc (400 mL). Theorganic layer was separated, dried over Na₂SO₄, filtered, and thesolvent was evaporated providing 28.9 g (98%) of the final product asclear colorless oil. ¹H NMR analysis showed the product was clean ofimpurities, and no further purification was attempted. Alternatively,N-t-Boc-5-amino-1-pentanol can be obtained from TCI America of Portland,Oreg.

Compound 2. Compound 2 was prepared by a procedure adopted from theliterature (Westerlind, U. et al. Glycoconj. J. 2004, 21, 227-241). To a500-mL one-neck round-bottom flask was added2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-galactopyranose 1 (12.8 g,32.8 mmol) followed by anhydrous CH₂Cl₂ (150 mL) and trimethylsilyltrifluoromethanesulfonate (14.3 mL, 79.2 mmol). This mixture was stirredat reflux overnight (ca. 18 h) under a flow of argon gas. The reactionmixture was cooled to 0° C. and treated with triethylamine (6.4 mL, 45.9mmol) for 30 min before being warmed to room temperature, then washedwith saturated aqueous NaHCO₃ (100 mL). The organic layer was separatedand dried over Na₂SO₄, filtered and evaporated providing crude oxazolineintermediate. To the crude oxazoline product was added anhydrous CH₂Cl₂(200 mL), N-t-Boc-5-amino-1-pentanol (10.0 g, 49.2 mmol) and 3 Åmolecular sieves (18.0 g, dried at 150° C. for >24 h). This mixture wasstirred at room temperature for 30 min under a blanket of argon gas.Trimethylsilyl trifluoromethanesulfonate (2.97 mL, 16.4 mmol) was addedto the reaction mixture, and the solution was stirred at roomtemperature overnight. The solution was cooled to 0° C. and treated withtriethylamine (3.2 mL, 23.07 mmol) for 30 min before being warmed toroom temperature. After the reaction reached room temperature themixture was filtered, and the mother liquor was evaporated providing thecrude product as brown oil which was dissolved in anhydrous pyridine(100 mL) and treated with acetic anhydride (36 mL, 38.2 mmol). Thismixture was stirred under an argon atmosphere at room temperatureovernight, then evaporated under vacuum yielding a brown liquid, whichwas dissolved in CH₂Cl₂ (200 mL). The solution was vigorously stirredwith a saturated aqueous NaHCO₃ solution (100 mL) and solid NaHCO₃ in anopen flask at room temperature to quench remaining Ac₂O and the organiclayer was separated. The aqueous layer was extracted with CH₂Cl₂ (1×200mL) and all organic layers were combined. The organic layers were washedwith saturated aqueous NaHCO₃ solution (1×100 mL), separated, dried overNa₂SO₄, filtered and evaporated providing the crude product as a brownoil which was then dissolved in CH₂Cl₂ (15 mL) and purified using columnchromatography (SiO₂, column size 7.5 cm ID×16.0 cm length,EtOAc:Hexanes 1:3 v/v for 500 mL, EtOAc:Hexanes 4:1 v/v for 500 mL, 100%EtOAc for 1.0 L, 10% MeOH in EtOAc v/v for 3.0 L). Product-containingfractions were pooled and evaporated under vacuum to a white solid whichwas further purified by trituration with ether to yield the desiredproduct as a white solid (5 g, 29%). ESI MS [M+H]⁺ m/z 533.4.

Compound 3. To a 100 mL round bottom flask was added Compound 2 (3.14 g,5.9 mmol) followed by trifluoroacetic acid (10 mL, TFA). The mixture wasstirred until all of the carbohydrate was completely dissolved, then theTFA was evaporated under vacuum to yield light yellow oil. To the oilyresidue was added diethyl ether (10 mL), the mixture was sonicated for2-5 min, and the supernatant was decanted. The trituration process wasrepeated (3×10 mL Et₂O), and the crude product was dried under vacuum toyield a white foam (3.2 g), which was used as described below.

Step 2.

Compound 7. To a 250 mL one-neck round-bottom flask was added Compound 6(3.37 g, 3.9 mmol, HPLC purified) followed by anhydrous CH₂Cl₂ (40.0mL), and triethylamine (2.17 mL, 15.6 mmol). This solution was stirredat 0° C. under a low flow of argon gas for 5 min before trifluoroaceticacid pentafluorophenyl ester (737 μL, 4.29 mmol) as added dropwise tothe reaction mixture. Then the mixture was warmed to room temperatureand was stirred at room temperature for 30 min.

The reaction progress was followed by TLC (SiO₂, CH₂Cl₂ and MeOH, 9:1v/v) by looking for the disappearance of the starting material(R_(f)=0.30) and the appearance of the PFP activated product(R_(f)=0.64). Once the starting material was consumed by TLC, the crudereaction was diluted with CH₂Cl₂ (300 mL) and the mixture was extractedusing NaHCO₃ (3×50 mL). The organic layer was separated, dried overNa₂SO₄, filtered and evaporated providing 3.9 g (97%) of the finalproduct as orange oil. All solvents and volatile reagents werethoroughly removed using high vacuum overnight before the crude productis carried on to the next synthetic step.

Compound 8. To a 100 mL one-neck round-bottom flask was added Compound 7(3.6 g, 3.5 mmol) followed by anhydrous acetonitrile (7.5 mL) andtriethylamine (1.46 mL, 10.5 mmol). The mixture was stirred under a flowof argon gas until all of the material was dissolved, then cooled to 0°C. with an ice bath. Deprotected amine 3 (1.81 g, 3.32 mmol) wasdissolved in anhydrous acetonitrile (7.5 mL), and the resulting solutionwas added to the reaction mixture at 0° C. dropwise over 5 min. Thereaction was allowed to warm to room temperature and was stirred at roomtemperature overnight. The solvents were evaporated using a rotaryevaporator, and the crude product was dried under high vacuum. Thereaction progress was followed by analytical HPLC by diluting thereaction mixture (5 μL) into CH₃CN (695 μL) and 50 μL of the dilutedmixture was analyzed by HPLC (10% CH₃CN for 2 min, then linear gradientfrom 10% to 60% CH₃CN over 20 min, total flow rate of 1.0 mL/min). Thedesired product had a retention time of 21.0 min.

The crude product was dissolved in MeOH (approximately 40 mL) andpurified in 2-mL aliquots using preparative reverse phase HPLC(Phenomenex, Luna 5 C18(2), 100 Å, 25.0 cm×21.2 mm, equipped with aSecurityGuard PREP Cartridge, C18 15×21.2 mm ID, CH₃CN/H2O, 30% CH₃CNfor 5 min, then linear gradient from 30% to 53% CH₃CN over 20 min, totalflow rate of 20.0 mL/min,). The desired product eluted between 22.0 and23.0 min. All the fractions containing the desired product werecombined, and the solvent was completely removed using a rotaryevaporator to yield 2.54 g (60%) of compound 8 after overnight dryingunder vacuum.

ESI MS: m/z 1277.6 ([M+H]⁺¹), 650.6 ([M+Na+H]⁺²), 658.5 ([M+K+H]⁺²),661.7 ([M+2Na]⁺²), 669.7 ([M+Na+K]⁺²), 677.5 ([M+2K]⁺²).

¹H NMR (CD3OD): δ 1.35 (t, 3H, J=7.5 Hz), 1.33-1.62 (m, 6H), 1.88 (s,3H), 1.93 (s, 3H), 1.95 (s, 3H), 2.03 (s, 3H), 2.15 (s, 3H), 2.32-2.56(m, 6H), 3.15-3.25 (m, 2H), 3.25-3.42 (m, 6H), 3.50-3.70 (m, 44H),3.97-4.20 (m, 4H), 4.55 (d, 1H, J=8.4 Hz), 5.05 (dd, 1H, J₁=11.4 Hz,J₂=3.4 Hz), 5.33 (dd, 1H, J₁=3.4 Hz, J₂=0.9 Hz).

Example 3. Synthesis of PolymerNagC5N-PEG-_(0.6)-[PEGMA4-5₈₀-PDSMA₁₀-BPAM₁₀]_(6.4)-b-[D25-B₅₀-P25]_(6.3)(P1) Example 3.1. Synthesis of Macro-CTA C1

PEGMA4-5 (0.675 g, 2.25 mmoles), PDSMA (0.072 g, 0.282 mmoles), BPAM(0.077 g, 0.282 mmoles), Nag(OAc4)C5N-PEG_(0.6K)-CTA (Compound 8) (0.090g, 0.0704 mmoles; 1:40 CTA:Monomers), AIBN (0.578 mg, 0.00252 mmoles;CTA:AIBN 20:1) and DMF (1.65 g) were introduced under nitrogen in asealed vial. The mixture was degassed by bubbling nitrogen for 30minutes, and the reaction was allowed to proceed at 68° C. with rapidstirring for 2 hours. The reaction was stopped by placing the vial inice and exposing the mixture to air. The polymer was purified bydialysis against methanol for 24 hours (Spectrum Labs, Spectra/PorDialysis Membrane MWCO: 2000), followed by removal of solvents undervacuum. The resulting Macro-CTA was dried under vacuum for 6 hours. Thestructure and composition of the purified polymer were verified by ¹HNMR, which also confirmed the absence of signals corresponding to vinylgroups of un-incorporated monomers. Purity of the polymer was confirmedby GPC analysis. M_(n,GPC)=7.7 kDa, dn/dc=0.05700, PDI=1.28.

Example 3.2. Synthesis of Polymer P1

BMA (0.246 g, 1.73 mmoles), PAA (0.099 g, 0.87 mmoles), DMAEMA (0.136 g,0.87 mmoles), MacroCTA C1 (0.113 g, 0.0147 mmoles; 1:236 CTA:Monomers),AIBN (0.241 mg, 0.00147 mmoles; CTA:AIBN 10:1) and DMF (0.615 g) wereintroduced in a vial. The mixture was degassed by bubbling nitrogen intothe mixture for 30 minutes, and then allowed to react for 10 hr at67-68° C. The reaction was stopped by placing the vial in ice andexposing the mixture to air. The polymer was purified by dialysis fromacetone/DMF 1:1 into hexane/ether 75/25 (three times). The resultingpolymer was dried under vacuum for at least 8 hours. The structure andcomposition of the purified polymer were verified by ¹H NMR, which alsoconfirmed the absence of signals corresponding to vinyl groups fromun-incorporated monomers. GPC analysis: M_(n)=13.996 kDa,dn/dc=0.056505, PDI=1.26.

The acetyl groups were removed by treatment of the polymer with sodiummethoxide (6 equivalents) in anhydrous methanol/chloroform under anatmosphere of argon at room temperature for 1.0 hour. The polymer wascapped with 2,2′-dipyridyl disulfide (2 equivalents relative to pyridyldisulfide residues in the polymer) at room temperature for 1.0 hourunder a flow of argon gas. After the capping the reaction was dilutedwith MeOH and filtered. The filtrate was transferred to a dialysismembrane with a 2000 g/mol molecular weight cut off (Spectrum Labs,Spectra/Por Dialysis Membrane MWCO: 2000) and dialyzed against MeOH over24 hours followed by dialysis against water. The solvent was evaporated,and the polymer was dried under vacuum.

Example 4. Synthesis of PolymerNagC5N-PEG_(0.6)-[PEGMA4-5₈₀-PDSMA₁₀-BPAM₁₀]_(7.2)-b-[D₂₅-B₅₀-P₂₅]_(6.1)(P2) Example 4.1. Preparation of MacroCTA C2

MacroCTA C2 was prepared as described in Example 3.1 starting fromPEGMA4-5 (8.083 g, 27.0 mmoles), PDSMA (0.860 g, 3.37 mmoles), BPAM(0.921 g, 3.37 mmoles), Nag(OAc4)C5N-PEG_(0.6K)-CTA (Compound 8) (1.076g, 0.842 mmoles; 1:40 CTA:Monomers), AIBN (6.914 mg, 0.0421 mmoles;CTA:AIBN 20:1) and DMF (19.73 g). Polymerization time was 2 hr 55 min.GPC: M_(n)=8.500 kDa; PDI˜1.23; dn/dc=0.5780

Example 4.2. Preparation of Polymer P2

Extension of MacroCTA C2 by RAFT polymerization was carried out asdescribed in Example 3.1 using BMA (0.553 g, 3.89 mmoles), PAA (0.226 g,1.98 mmoles), DMAEMA (0.311 g, 1.98 mmoles), MacroCTA C2 (0.560 g,0.0659 mmoles; 1:118 CTA:Monomers), AIBN (1.082 mg, 0.00659 mmoles;CTA:AIBN 10:1) and DMF (1.37 g+0.69 g). Polymerization was stopped after5 hours, and the product was purified by dialysis from Acetone/DMF 1:1into hexane/ether 75/25 (three times). GPC: dn/dc=0.053188; M_(n)=14.7kDa; PDI=1.31. The acetyl groups were removed with NaOMe as described inExample 3.2.

Example 5. Synthesis of PolymerNagC5N-PEG_(0.6)-[PEGMA4-5₈₀-PDSMA₁₀-BPAM₁₀]_(7.2)-b-[D₂₅-B₅₀-P₂₅]_(10.8)(P3)

MacroCTA C2 (Example 4) was extended by RAFT polymerization as describedin Example 3.2 using BMA (0.197 g, 1.39 mmoles), PAA (0.079 g, 0.69mmoles), DMAEMA (0.109 g, 0.69 mmoles), Macro-CTA (0.100 g, 0.0118mmoles; 1:236 CTA:Monomers), AIBN (0.193 mg, 0.00118 mmoles; CTA:AIBN10:1) and DMF (0.492 g) for 4.5 hours, and the product was purified bydialysis from Acetone/DMF 1:1 into hexane/ether 75/25 (three times).GPC: dn/dc=0.053160; Mn=19.3 kDa; PDI=1.39. The acetyl groups wereremoved with NaOMe as described in Example 3.2.

Example 6. Synthesis of PolymerPEG-_(0.6)-[PEGMA4-5₈₀-PDSMA₁₀-BPAM₁₀]_(6.7)-b-[D₂₅-B₅₀-P₂₅]_(6.2) (P4)Example 6.1 Preparation of MacroCTA C4

Macro-CTA C4 was prepared as described in Example 3 starting withPEGMA4-5 (5.128 g, 17.1 mmoles), PDSMA (0.546 g, 2.14 mmoles), BPAM(0.584 g, 2.14 mmoles), PEG_(0.6K)-CTA (Compound 6) (0.461 g, 0.534mmoles; 1:40 CTA:Monomers), AIBN (4.385 mg, 0.0267 mmoles; CTA:AIBN20:1) and DMF (12.52 g); reaction time was 1 hr 40 min. GPC: Mn=7.50kDa; PDI˜1.20; dn/dc=0.053910.

Example 6.2 Preparation of Polymer P4

Synthesis and purification of Polymer P4 was carried out as described inExample 3.2 using BMA (1.656 g, 11.6 mmoles), PAA (0.676 g, 5.92mmoles), DMAEMA (0.931 g, 5.92 mmoles), MacroCTA C4 (1.5 g, 0.197mmoles; 1:118 CTA:Monomers), AIBN (3.241 mg, 0.0197 mmoles; CTA:AIBN10:1) and DMF (4.16 g+2.08 g). GPC: dn/dc=0.050; M_(n)=13.8 kDa;PDI=1.1.

Example 7. Synthesis of PolymerNagC5N-PEG_(0.6)-[PEGMA4-5₈₀-PDSMA₁₀-BPAM₁₀]_(6.6)-b-[D₂₅-B₅₀-P₂₅]_(114.7)(P5) Example 7.1 Preparation of MacroCTA C5

MacroCTA C5 was synthesized as described in Example 3.1 starting fromPEGMA4-5 (0.5 g, 1.67 mmoles), PDSMA (0.053 g, 0.208 mmoles), BPAM(0.057 g, 0.208 mmoles), Nag(OAc4)C5N-PEG_(0.6K)-CTA (Compound 8)(0.0665 g, 0.0521 mmoles; 1:40 CTA:Monomers), AIBN (0.428 mg, 0.0026mmoles; CTA:AIBN 20:1) and DMF (1.22 g). Polymerization time was 2 hr 30min. GPC: Mn=7.85 kDa; PDI=1.18; dn/dc=0.066.

Example 7.2 Preparation of Polymer P5

Synthesis and purification of Polymer P5 was carried out as described inExample 3.2 using BMA (0.62 g, 4.36 mmoles), PAA (0.249 g, 2.18 mmoles),DMAEMA (0.342 g, 2.18 mmoles), MacroCTA C5 (0.189 g, 0.0242 mmoles;1:360 CTA:Monomers), AIBN (0.398 mg, 0.00242 mmoles; CTA:AIBN 10:1) andDMF (1.55 g). Polymerization was allowed to proceed for 10 hrs. GPC:dn/dc=0.063851; M_(n)=22.5 kDa; PDI=1.41. Deprotection was carried outas described in Example 3.2.

Example 8. Synthesis of PolymerNagC5N-PEG_(0.6)-[PEGMA4-5₈₀-PDSMA₁₀-BPAM₁₀]_(3.5)-b-[D₂₅-B₅₀-P₂₅]_(16.3)(P6) Example 8.1 Preparation of MacroCTA C6

Macro-CTA C6 was synthesized as described in Example 3.1 starting fromPEGMA4-5 (1.503 g, 5.00 mmoles), PDSMA (0.160 g, 0.626 mmoles), BPAM(0.171 g, 0.626 mmoles), Nag(OAc4)C5N-PEG_(0.6K)-CTA (Compound 8) (0.500g, 0.391 mmoles; 1:40 CTA:Monomers), AIBN (3.213 mg, 0.0196 mmoles;CTA:AIBN 20:1) and DMF (3.668 g); reaction time was 1 hr 45 min. GPC:M_(n)=4.8 kDa; PDI=1.19; dn/dc=0.061481.

Example 8.2 Preparation of Polymer P6

Synthesis and purification of Polymer P6 was carried out as described inExample 3.2 using BMA (0.218 g, 1.54 mmoles), PAA (0.089 g, 0.781mmoles), DMAEMA (0.123 g, 0.781 mmoles), MacroCTA C6 (0.125 g, 0.0260mmoles; 1:118 CTA:Monomers), AIBN (0.428 mg, 0.00260 mmoles; CTA:AIBN10:1) and DMF (0.830 g). Polymerization was allowed to proceed for 4 hrsand 50 min. GPC: dn/dc=0.05812; M_(n)=11.1 kDa; PDI=1.38. Deprotectionwas carried out as described in Example 3.2.

Example 9. Synthesis of PolymerNagC5N-PEG_(0.6)-[PEGMA4-5₈₆-PDSMA₁₄]_(3.82KDa)-[BMA₄₅-PAA₁₅-DMAEMA₄₀]_(5.98KDa)(P⁷) Example 9.1 Preparation of Nag(OH)C5N-PEG_(0.6K)-CTA (Compound 8a)

Nag(OH)C5N-PEG_(0.6)K-CTA (Compound 8a) was prepared in a similar mannerto the Nag(OAc4)C5N-PEG_(0.6K)-CTA in Example 2 (Compound 8) except thatcompound 3 in Example 2 is replaced by the unprotected sugar compound ofcompound 3a and the coupling reaction between compound 6 of Example 2and compound 3 of Example 2 has been modified as shown below forcompounds 6a and 3a.

Compound 3a is prepared as follows from compound 3b.

To a 250 mL one-neck round-bottom flask was added compound 3b (1.86 g,3.5 mmol) followed by 4M HCl in dioxane (30 mL). This mixture wasstirred and sonicated until all of the sugar was completely dissolved.Then the mixture was evaporated on a rotary evaporator providing an oilyresidue. To completely remove all HCl gas the compound was dissolved indioxane (30 mL) and solvents removed by rotary evaporation. The solventexchange process was preformed a total of 3 times to completely removeall HCl. Then the flask was put under high vacuum for >30 min providinga white foam solid. The crude compound was dissolved in anhydrous MeOH(25 mL) and treated with 0.5 M sodium methoxide solution in MeOH (5.80g, 7.175 mL, 3.59 mmol, 1.025 eq, measured by weight to ensure accuracyof addition). The first equivalent of NaOMe is used to de-protonate thequaternary amine salt liberating the free amine. Only a slight excess ofNaOMe beyond one equivalent (i.e., 0.025 eq, 0.09 mmol) is needed tofacilitate the acetyl deprotection. Once NaOMe is added the mixture isthen stirred under a flow of argon overnight at room temperature.Reaction progress was monitored by LCMS using Agilent Q-TOF LiquidChromatography Mass Spectrometer by dissolving the product in MeOH atca. 1.0 μg/mL. The LC used a C18 UPLC column (Agilent Eclipse Plus C18,catalog number 959757-902, 1.8 μm, 2.1 mm×50 mm, column at roomtemperature, CH₃CN/H₂O containing 0.1% formic acid, isocratic gradientat 5% CH₃CN for 1 min, then linear gradient from 5% to 90% CH₃CN over 4min, total flow rate of 0.4 mL/min). The desired product elutes between0.4-0.5 min using the above HPLC conditions while the crude intermediateproduct (i.e., Boc removed with acetyls still present) elutes between2.0-2.2 min. Once the sugar was fully de-protected the catalytic NaOMe(0.09 mmol) is quenched by adding a slight excess of acetic acid (10 μL,0.175 mmol) to the reaction mixture. Then all solvents are removed byevaporating on a rotary evaporator. This process yielded 1.1 g (100%) ofthe final product as a white solid. The final product was characterizedusing a 400 MHz 1H NMR with CD₃OD as solvent and all spectra wereconsistent with the desired product compound 3a.

Nag(OH)C5N-PEG_(0.6)K-CTA (Compound 8a) was prepared as follows.Compound 6a was prepared as in Example 2 (Compound 6).

To a 250 mL one-neck round-bottom flask was added compound 6a (3.17 g,3.68 mmol) followed by anhydrous acetonitrile (10 mL). In a separateflaks the compound 3a (1.07 g, 3.5 mmol) was dissolved in anhydrous DMF(10 mL). Once compound 3a was partially dissolved as a milky whitesuspension the solution was transferred to a 100 mL addition funnel. Inanother flask was added PyBOP (2.0 g, 3.85 mmol) and anhydrous DMF (10mL). The PyBOP/DMF solution was taken up into a 20 mL syringe. Then all3 solutions (compound 6a/CH₃CN, compound 3a/DMF, and PyBOB/DMF) werecombined simultaneously and as fast as possible while the reactionsolution was vigorously stirred. Once the additions were complete thereaction was treated with N,N-diisopropylethylamine (1.22 mL, 7.0 mmol)and the solution was stirred at room temperature under a flow of argongas for 30 min. The reaction progress was determined using Agilent Q-TOFLiquid Chromatography Mass Spectrometer by dissolving the crude reaction(1.0 μL) into MeOH (1.0 mL) and injecting 1.0 μL (FIGS. 1-2). The LCused a C18 UPLC column (Agilent Eclipse Plus C18, catalog number959757-902, 1.8 μm, 2.1 mm×50 mm, column at room temperature, CH₃CN/H₂Ocontaining 0.1% formic acid, isocratic gradient at 5% CH₃CN for 1 min,then linear gradient from 5% to 90% CH₃CN over 4 min, total flow rate of0.4 mL/min). The desired product elutes between 3.0-3.1 min using theabove HPLC conditions. The sugar starting material (i.e., compound 3a)was not detected on the mass spec analysis after the reaction wasstirred at room temperature for 30 min. Mass spec analysis confirms thepresence of compound 8a [M+Na]⁺¹=1173.5207 m/z; [M+H]⁺¹=1151.5397 m/z).

After reacting for 30 min the crude reaction mixture of compound 8a wasdiluted by the addition of H₂O (25 mL) and purified using C18preparative reverse phase HPLC by Shimadzu (Phenomenex, Luna 5 C18(2),part number 00G-4252-P0-AX, 100 Å, 25.0 cm×21.2 mm, with a SecurityGuardPREP Cartridge, C18 15×21.2 mm ID, part number AJ0-7839, CH₃CN/H₂O with0.01% TFA, isocratic gradient at 5% CH₃CN for 5 min, then lineargradient from 5% to 50% CH₃CN over 17 min, then 50% to 53% CH3CN over 3min, total flow rate of 20.0 mL/min, column at room temperature). 2.0 mLof the crude compound dissolved in DMF/H₂O (ca. 75 mg/mL) were injectedeach HPLC run. Using the HPLC purification conditions above the desiredproduct compound 8a eluted between 21.5 and 22.5 min. All the fractionscontaining the desired product were combined and the water/CH3CN solventwas completely removed using a rotary evaporator then high vacuumovernight. The combined yield of the final product after HPLCpurification and overnight high vacuum produced 3.05 g (76%) of thedesired product as a bright orange solid. ¹H NMR analysis was consistentwith the presence of the desired product compound 8a.

Example 9.2 Preparation of MacroCTA C7

AIBN/DMF (21.93 g of 1.05603 mg/g ABIN in DMF) was added toNag(OH)C5N-PEG_(0.6K)-CTA (synthesized as described in Example 9.1,compound 8a) (3.075 g; 2.6705 mmol) in a 40 ml reaction vessel and mixedto dissolve the CTA. DMF was then added until the total weight of DMFwas 24.9627 g. To the resulting solution was added PEGMA (11.18 g,37.2621 mmoles, filtered through aluminum oxide (activated, basic,Brockmann I) and PDSMA (1.1211 g, 4.1393 mmoles). The resulting solutionwas mixed and then transferred to a sealed 50 mL round bottom flaskequipped with a magnetic stir bar. The resulting solution wasde-oxygenated by bubbling nitrogen into the solution for 50 min on ice.The flask was moved to room temperature for 4 min and then placed in anoil bath pre-heated to 68° C. for 1 hour 42 minutes (stir speed was setat 350 rpm). The reaction was stopped by placing the vial in ice andexposing the mixture to air. The reaction solution was diluted withMeOH, transferred to dialysis membranes (Spectrum Labs, SpectrumSpectra/Por 6 Dialysis Membrane Tubing MWCO: 2000) and dialyzed againstMeOH (6×4000 mL) for 6 days. Samples were taken for LC-MS, GPC and ¹HNMR analyses. After dialysis, the solvent was removed under reducedatmosphere followed by high vacuum to afford 2.45 g of polymer. LC-MSanalysis indicated no residual CTA peak. ¹H NMR, which also confirmedthe absence of signals corresponding to vinyl groups of un-incorporatedmonomers. Purity of the polymer was confirmed by GPC analysis.M_(n,GPC)=4.97 KDa, PDI=1.12, dn/dc=0.06469, PDI=1.12. Alternatively,macro-CTA C7 can be synthesized as described in Example 3.1 startingfrom PEGMA4-5 and PDSMA quantities described above.

Example 9.3. Synthesis of Polymer P7

AIBN/DMF solution (7.0225 g; 1.10468 mg/g AIBN in DMF) was added tomacro-CTA C7 (2.350 g) in a 40 mL reaction vessel; the sample was mixedto dissolve the macro-CTA. DMF was then added until the total weight ofDMF was 15.05 g. BMA (3.967 g, filtered through aluminum oxide(activated, basic, Brockmann I), PAA (1.6217 g) and DMAEMA (2.237 g,filtered through aluminum oxide [activated, basic, Brockmann I]) wereadded to the resulting solution and the solution was mixed. The mixturewas vortexed for several minutes to give a homogeneous stock solutionand transferred to a sealed 50 mL round bottom flask equipped with amagnetic stir bar. The mixture was then cooled to 0° C. using an icebath and maintained at 0° C. while degassed by vigorously bubblingnitrogen inside the solution for 55 minutes. The flask septa was placedinto an oil bath pre-heated to 61° C. (stirring speed was 350) andallowed to stir for 4 hours 30 minutes. The reaction was stopped byplacing the vial in ice and exposing the mixture to air. The reactionwas then diluted with acetone (roughly the same volume of acetone as theDMF used in the reaction vial) and precipitated into a stirred mixtureof ether/hexanes (1:3 v/v) in a 50 mL centrifuge tube once and then intoa large beaker with 600 mL ether/hexanes (1:3 v/v). The polymerprecipitate was isolated and dissolved with MeOH, transferred to threeindividual dialysis membranes (Spectrum Labs, Spectrum Spectra/Por 6Dialysis Membrane Tubing MWCO: 2,000) and dialyzed against methanol(5×4000 mL) for 4 days. After the dialysis against methanol, it wasdialyzed against nanopure water using the same membrane (×6, waterchanged every hour). When the dialysis was complete, the solution wastransferred to tared vials and treated with liquid nitrogen before beinglyophilized for 5 days to afford 3.46 g of the final product. The finalproduct was analyzed by UV/vis, NMR, GPC and HPLC equipped with RIdetector (for batch dn/dc). Analysis of the polymer by ¹H-NMR indicateda polymer with no vinyl groups remaining and the presence of PDSMA. TheNMR is consistent for proposed structure. GPC results: Mn=10.936 KDa,PDI=1.30, dn/dc=0.057867.

Example 10. Synthesis of PolymerNAG-PEG_(0.6)-[PEGMA₁₀₀]_(3.5k)-[BMA₄₉-PAA₁₀-DMAEMA₃₃-PDSMA₈]_(7.1k)(P8) Example 10.1 Preparation of MacroCTA C8

To a 20 mL reaction vial was added to Nag(OH)C5N-PEG_(0.6K)-CTA(synthesized as described in Example 9.1, compound 8a) (794.6 mg, 0.6922mmol, CTA) followed by a solution of AIBN (5.0438 g solution dissolvedin DMF at a concentration of 1.1268 mg/g, 5.68 mg AIBN, 0.03461 mmol,2,2′-azobis(2-methylpropionitrile), compound recrystallized from MeOH)then an additional amount of DMF (432.2 mg) was added bringing the totalamount of DMF used in this reaction to 5.4760 g. This solution was mixedand vortexed for several minutes until all of the CTA was completelydissolved. Once all the CTA was completely dissolved PEGMA (3219.3 mg,10.730 mmol, poly(ethylene glycol) methyl ether methacrylate withaverage M_(n)=300 g/mol, inhibited with 100 ppm MEHQ and 300 ppm of BHTinhibitors, Aldrich part number 447935-500 mL, inhibitors removed bypassing the neat monomer through a plug of Al₂O₃, was added to thereaction vial. This mixture was stirred for several minutes. Thereaction vial was partially sealed and cooled to 0° C. using an ice bathwhile the mixture was degassed by vigorously bubbling nitrogen for 30minutes with magnetic stirring of the reaction solution. Then the vialwas completely sealed and placed into a heater block. The stirring speedwas set at 300 rpm, the thermometer was set at 68° C. and was maintainedat this temperature during the entire process. The reaction was left tostir at 68° C. for 1 hours and 47 minutes. After the reaction iscomplete it was quenched by opening the vial and then placing thereaction vial in ice exposing the mixture to air. The reaction vial wasdiluted with MeOH (10 mL) and transferred to a dialysis membrane with a2000 g/mol molecular weight cut off (Spectrum Labs, Spectrum Spectra/Por6 Dialysis Membrane Tubing MWCO: 2000) and dialyzed against MeOH (3×4000mL) for 4 days. The dialysis solution was changed every day for 3iterations total. The polymer in the dialysis bag was analyzed accordingto the following procedure: A small aliquot of the dialysis solution(ca. 500-1000 μL) was withdrawn from the dialysis tubing and placed intoa tared vial. The solution was then evaporated using a rotaryevaporator. Once the solvents are removed the vial was transferred to ahigh vacuum line and placed under high vacuum. The compound is dried for<15 min. Once the vial weight is constant then the compound wasdissolved immediately in DMF with 1% weight LiBr solution. The finalconcentration of the polymer was approximately 8 mg/mL in DMF with 1% wtLiBr (DMF measured by weight then converted to volume). A 20 kDapolystyrene standard (Fluka, part number 81407-1G) dissolved in DMF with1% wt LiBr at a concentration of roughly 3 mg/mL (DMF measured by weightthen converted to volume) is then injected (100 μL) on the GPC followedby the polymer sample of interest (60, 80, 100, and 120 μL). Once thefinal GPC analysis is determined then the dialysis solution wastransferred to a 40 mL reaction vial then the solvents were removedusing a rotary evaporator. Then the material was place on a high vacuumline (pressure <0.5 torr) for >24 hours. This process provided 682.9 mgof the final product. The final product is then analyzed by NMR and GPC.The final product was stored at room temperature under high vacuum. TheNMR is consistent for proposed structure. GPC results: Mn=4.600,dn/dc=0.053354.

Example 10.2. Synthesis of Polymer P8

To a 40 mL reaction vial was added macro-CTA C8 (682.1 mg, 0.148 mmol)followed by a solution of AIBN (2.2338 g solution dissolved in DMF at aconcentration of 1.0927 mg/g, (2.44 mg AIBN, 0.0148 mmol,2,2′-azobis(2-methylpropionitrile), compound recrystallized from MeOH)then an additional amount of DMF (2.6163 g) was added bringing the totalamount of DMF used in this reaction to 4.8501 g. This solution was mixedand vortexed for several minutes until all of the CTA was completelydissolved. Once all the CTA was completely dissolved then BMA (1.1849 g,8.314 mmoles, purified by passing the neat monomer through a plug ofAl₂O₃, butyl methacrylate, d-0.894 g/mL), PAA (488.0 mg, 4.231 mmoles,unpurified 2-propylacrylic acid, d-0.951 g/mL), DMAEMA (661.8 mg, 4.231mmoles, purified by passing the neat monomer through a plug of Al₂O₃,2-(dimethylamino)ethyl methacrylate, d-0.933 g/mL), and PDSMA (227.0 mg,0891 mmol). This mixture was mixed for several minutes. The reactionmixture was then transferred to a brand new 20 mL reaction vialcontaining a magnetic stir bar. The reaction vial was partially sealedand cooled to 0° C. using an ice bath while the mixture was degassed byvigorously bubbling nitrogen for 30 minutes with magnetic stirring ofthe reaction solution. The vial was then completely sealed and placedinto a heater block. The stirring speed was set at 300, the thermometerwas set at 62° C. The reaction was left to stir at 62° C. for 5 hoursand 50 minutes. After the reaction is complete it was quenched byopening the vial and then placing the reaction vial in ice exposing themixture to air. The reaction solution was then diluted with acetone (˜5mL, roughly the same volume of acetone as the DMF used in the reactionvial) and precipitated into a stirred mixture of Et₂O/hexanes (1000 mL,1:4 v/v) in a glass beaker. After the polymer had settled to the bottom(ca. 15 min) the solvents were decanted off. The precipitated polymerdissolved in MeOH was transferred into dialysis membranes with a 2000g/mol molecular weight cut off (Spectrum Labs, Spectrum Spectra/Por 6Dialysis Membrane Tubing MWCO: 2000) and dialyzed against MeOH (3×4000mL) for 3 days (72 h). The dialysis solution was changed every day for 3iterations total. After 3 days (72 h) dialysis against MeOH the dialysissolution is changed to nanopure H₂O and dialyzed against H₂O (5×4000 mL)for 5 hr. The dialysis solution was changed roughly every hour for 5iterations total. Upon completion of dialysis the solutions weretransferred to tared vials and frozen solid using a bucket of dry ice.Then the material was placed into the lyophilizer for >4 days totaldrying time. This process provided 1.0325 g of the final product. Thefinal product was then analyzed by NMR and GPC. Analysis of the polymerby ¹H-NMR indicated a polymer with no vinyl groups remaining and thepresence of PDSMA. The NMR is consistent for proposed structure. GPCresults: Mn=11.7 kDa, dn/dc=0.058046. The final product was stored inglass vials with rubber septum that were purged with argon and sealedwith parafilm. The vials were stored at −20° C.

Example 11. Synthesis of H₂N-Cys-Lys₍₁₀₎-OH (SEQ ID NO:101)

The cysteine-terminated oligolysine, NH2-CKKKKKKKKKK-COOH(H₂N-Cys-Lys₍₁₀₎-OH) (SEQ ID NO:101), was synthesized on a solid Wangsupport following standard Fmoc/tBu chemistry by manual synthesis.Fmoc-protected amino acids were activated using HBTU and DIPEA ascoupling agents. Coupling was verified by a negative ninhydrin assay.Fmoc was removed from coupled residue with treatment by a 20% solutionof piperidine in DMF. CK10 was cleaved from resin by treating the solidsupport with TFA/dimethoxybenzene/TIPS (95:5:2.5:2.5, v/v/v/v). Cleavedpeptides were then precipitated in cold ether, dissolved in methanol andreprecipitated in cold ether. Peptides were purified by semi-preparativeRP-HPLC using a Jupiter Luna C18 300A column 250×21.0 mm (Phenomenex,Torrance, Calif.). Fractions were pooled and re-analyzed by RP-HPLC andLC/MS and demonstrated a purity of greater than 95%.

The following additional peptides, prepared by automated synthesis, werepurchased:

(SEQ ID NO: 103) H₂N-Cys-Lys₍₁₀₎-NH₂ (SEQ ID NO: 102) H₂N-Cys-Arg₍₁₀₎-OH(SEQ ID NO: 104) H₂N-Cys-Arg₍₁₀₎-NH₂.

Example 12. Synthesis of PolymerNAG-PEG_(0.6)-[PEGMA₉₀-PDSMA₁₀]_(5.3kDa)-b-[BMA₅₅-PAA₁₀-DMAEMA₃₅]_(6.7kDa)Conjugated to Poly-Lysine Peptide (P9)

A stock solution ofNAG-PEG_(0.6)[PEGMA₉₀-PDSMA₁₀]_(5.3kDa)-b-[BMA₅₅-PAA₁₀-DMAEMA₃₅]_(6.75)kDa(prepared as in Example 9.3 but with differing monomer incorporationrates) was prepared in MeOH at 300 mg/mL and briefly degassed with amoderate flow of argon for 2-3 min. Concurrently, a stock solution ofpoly-lysine peptide (prepared as described in Example 11) was preparedin MeOH at 50 mg/mL and briefly degassed with a moderate flow of argonfor 2-3 min. The polymer solution containingNAG-PEG_(0.6)-[PEGMA₉₀-PDSMA₁₀]_(5.3kDa)-b-[BMA₅₅-PAA₁₀-DMAEMA₃₅]_(16.75kDa)(440.0 μL, 132.0 mg polymer, 0.0116 mmol polymer, 0.0145 mmol PDS) wasthen transferred to a new 40 mL reaction vial. To the stirred vialcontainingNAG-PEG_(0.6)-[PEGMA₉₀-PDSMA₁₀]_(5.3kDa)-b-[BMA₅₅-PAA₁₀-DMAEMA₃₅]_(6.75kDa)was rapidly added an aliquot of the peptide solution (706.0 μL, 35.3 mgpeptide, 0.0139 mmol peptide). The mixture was covered with anatmosphere of argon, sealed, and then allowed to react for 25 min. Next,the reaction was opened and treated with N-maleoyl-β-alanine (16 μL of astock solution prepared in MeOH at 50 mg/mL, 8.0 mg, 0.047 mmol) to capany unreacted sulfhydryl groups. This capping procedure was allowed tostir at room temperature for another 25 min. Then the solution wasdiluted with MeOH (20 mL), transferred to a dialysis membrane (MWCO3500) and dialyzed in MeOH (1×4000 mL) for 2 hours. The dialysissolution was changed and the product was dialized overnight in MeOH(1×4000 mL) for 16 hours. The next day the dialysis solution was changeto 20 mM acetate buffer at pH 4.5, and dialyzed for 6 hours. Then thesolution was replaced with nanopure water (1×4000 mL, no buffer) andleft to dialyze overnight. The next day multiple changes of the nanopureH₂O dialysis solution were made (3×4000 mL) over a 4 hour period. Thedialysis solution was changed roughly every hour for 3 iterations totalto completely remove excess NaOAc salts. When the dialysis is completethe solution was transferred to tared vials and frozen solid using abucket of dry ice. Then the material was placed into the lyophilizerfor >4 days total drying time. This process provided 123 mg of the finalproduct. The final product was then analyzed by NMR. NMR analysis of theconjugate showed that essentially all of the pyridyl disulfide (PDS)groups in the conjugation block were removed. This implies nearlycomplete substitution of the PDS groups for poly-Lysine peptide and afully loaded polymer-peptide conjugate. The final product was stored inglass vials with rubber septum that were purged with argon and sealed.The vials were stored at −20° C.

Example 13. Synthesis of PolymerNAG-PEG_(0.6)-[PEGMA₁₀₀]_(35k)-[BMA₄₉-PAA₁₀-DMAEMA₃₃-PDSMA₈]_(7.1k).Conjugated to Poly-Lysine Peptide (P10)

To a solution ofNAG-PEG_(0.6)-[PEGMA₁₀₀]_(3.5k)-[BMA₄₉-PAA₁₀-DMAEMA₃₃-PDSMA₈]_(7.1)k(200 mg) in MeOH (665 μl) (prepared as described in Example 10.2) wasadded a solution of poly-Lysine peptide (H₂N-Cys-Lys(10)-OH (8 TFA)prepared as described in Example 11) (51.7 mg) in MeOH (665 μl). Basedon polymer PDS content, the amount of peptide added represents 0.5 eqper PDS group. The resulting solution was stirred at ambient temperaturefor 25 min. The solution was diluted with MeOH (20 fold, 26.5 ml), anddialyzed against MeOH (MWCO 3500). MeOH was exchanged after 2 hrs anddialysis continued for an additional 16 hrs. The solution was furtherdialyzed against 20 mM acetate buffer with 1 mM EDTA, pH 4.0 for 6 hrs.The dialysis buffer was exchanged, and dialysis continued for 16 hrs.Following dialysis, the solution was transferred to a 20 ml vial, frozenwith liquid nitrogen, and lyophilized for 3 days to afford 150 mgpolymer as a white powder. The final product was then analyzed by NMR.NMR analysis of the conjugate showed that essentially all of the pyridyldisulfide (PDS) groups in the conjugation block were removed. Thisimplies nearly complete substitution of the PDS groups for poly-Lysinepeptide and a fully loaded polymer-peptide conjugate. Alternatively, theNAG-PEG_(0.6)-[PEGMA₁₀₀]_(3.5k)-[BMA₄₉-PAA₁₀-DMAEMA₃₃-PDSMA₈]_(7.1k)poly-Lysine peptide conjugate of this Example 13 can be prepared usingthe methods previously described in Example 12.

Example 14. Determining Monomer Incorporation Within Individual Blocksof a Polymer During Polymer Synthesis

The amount of a given monomer within a given polymer block, typicallythe polymer block to which the oligonucleotide or peptide is conjugated,of the polymers exemplified and claimed herein has been determined bythe following procedure. Samples taken before and after thepolymerization reaction (i.e., T₀ (time zero) and T_(f)(time final)) areanalyzed by analytical HPLC to determine the extent of monomerconsumption and/or monomer incorporation.

The initial monomer amounts in the polymerization reaction (time 0, T₀)are determined by sampling the polymerization reaction solution prior tonitrogen or argon purge. A (20 μL) sample of the reaction solution iswithdrawn from the reaction solution and diluted into 180 μL of Methanol(MeOH). A portion of the resulting solution (10 μL) is further dilutedinto 590 μL MeOH, to afford a test sample with an overall dilution of1:600 (from the polymerization reaction) for analysis by analyticalHPLC.

Upon completion of the polymerization reaction a time final (T_(f))sample is prepared analogous to the T₀ sample described above.

Analytical HPLC analysis of the T₀, and T_(f) samples are performedusing a C18 Phenomenex 5μ 100 Å 250×4.6 mm×5 micron (Part #00G-4252-E0)Luna column with guard column heated to 30° C. Three independentdilutions for each time point (i.e., T₀, and T_(f)) are prepared andanalyzed for each time point. A 10 μl of sample is injected onto thecolumn and eluted with the following gradient. Hold an isocratic eluentof 5% acetonitrile/water with 0.1% TFA for 2 minutes. Switch to a lineargradient from 5% to 95% acetonitrile over 25 minutes. Hold an isocraticeluent of 95% acetonitrile for 5 minutes. Return to 5% acetonitrile over0.01 minutes. Hold the isocratic eluent of 5% acetonitrile/water with0.1% TFA for 5 minutes.

The following methodology is used to calculate the % incorporation of agiven monomer:

To calculate the % incorporation of a given monomer:

-   -   a. Calculate the consumption of individual monomers in the        reaction (monomer % consumption):        =(1−(T _(f) monomer peak area/T ₀ monomer peak area)×100.    -   b. Calculate the molar fraction consumed of the individual        monomers based on monomer input percent        =(Monomer % conversion (calculated in step (a)        above)×0.01)×monomer feed %    -   c. Total monomer consumption in the polymerization reaction and        overall percent conversion:        -   i. Total monomer consumption=sum of molar fraction consumed            for the individual monomers calculated in step (b) above.        -   ii. Overall % conversion=Average of total monomer            consumption (calculated in step (c)(i) above) from the 3            individual preparations×100.    -   d. Calculate the percent monomer incorporation for each monomer        in the polymer        -   i. =(Monomer molar fraction consumed (step (b) above)/total            monomer consumed (step (c)(i) above)×100.        -   ii. Average percent monomer incorporation for the 3            independent preparations.

Example 15. Determining Monomer Incorporation Within Individual Blocksof a Polymer During Polymer Synthesis

The amount of a given monomer within a given polymer block, typicallythe polymer block containing PAA, BMA and DMAEAMA, of the polymersexemplified and claimed herein has been determined by the followingprocedure. Samples taken before and after the polymerization reaction(i.e., T₀ (time zero) and T_(f)(time final)) are analyzed by analyticalHPLC to determine the extent of monomer consumption and/or monomerincorporation.

The initial monomer amounts in the polymerization reaction (time 0, T₀)are determined by sampling the polymerization reaction solution prior tonitrogen purge. A (20 μL) sample of the reaction solution is withdrawnand diluted into 180 μL of 1,1,1,3,3,3-hexafluoro-2-propanol(HFIP)/Methanol (MeOH)/Nano-pure water (H₂O) (2:1:1, v/v) containing0.1% TFA. A portion of the resulting solution (10 μL) is further dilutedinto 590 μL of HFIP/MeOH/H₂O (2:1:1, v/v) containing 0.1% TFA, to afforda test sample with an overall dilution of 1:600 (from the polymerizationreaction) for analysis by analytical HPLC.

Upon completion of the polymerization reaction a time final (T_(f))sample is prepared analogous to the T₀ sample described above. A (20 μL)sample of the reaction solution is withdrawn and diluted into 180 μL of1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)/Methanol (MeOH)/Nano-pure water(H₂O) (2:1:1, v/v) containing 0.1% TFA. A portion of the resultingsolution (10 μL) is further diluted into 590 μL of HFIP/MeOH/H₂O (2:1:1,v/v) containing 0.1% TFA, to afford a test sample with an overalldilution of 1:600 (from the polymerization reaction) for analysis byanalytical HPLC.

Analytical HPLC analysis of the T₀, and T_(f) samples are performedusing a C18 Phenomenex 5μ 100 Å 250×4.6 mm×5 micron (Part #00G-4252-E0)Luna column with guard column heated to 30° C. Three independentdilutions for each time point (i.e., T₀, and T_(f)) are to be preparedand analyzed. A 10 μl of sample is injected onto the column and elutedwith the following gradient. Hold an isocratic eluent of 5%acetonitrile/water with 0.1% TFA for 10 minutes. Switch to a lineargradient from 5% to 15% acetonitrile over 10 minutes. Switch to a lineargradient from 15% to 95% acetonitrile over 20 minutes. Hold an isocraticeluent of 95% eluent acetonitrile for 5 minutes. Return to 5%acetonitrile over 0.01 minutes. Hold the isocratic eluent of 5%acetonitrile/water with 0.1% TFA for 5 minutes.

The following methodology is used to calculate the % incorporation of agiven monomer:

-   -   e. Calculate the consumption of individual monomers in the        reaction (monomer % consumption):        =(1−(T _(f) monomer peak area/T ₀ monomer peak area)×100    -   f. Calculate the molar fraction consumed of the individual        monomers based on monomer input percent        =(Monomer % conversion (calculated in step 3.4a)×0.01)×monomer        feed % (DMAEMA=0.25,PAA=0.25,BMA=0.50)    -   g. Total monomer consumption in the polymerization reaction and        overall percent conversion:        -   iii. Total monomer consumption=sum of molar fraction            consumed for the individual monomers calculated in (b).        -   iv. Overall % conversion=Average of total monomer            consumption (calculated in (c) (i) from the 3 individual            preparations×100    -   h. Calculate the percent monomer incorporation for each monomer        in the polymer        -   i. =(Monomer molar fraction consumed (calculated in (b)            above)/total monomer consumed (calculated in (c)(i)))×100        -   ii. Average percent monomer incorporation for the 3            independent preparations.

Example 16. Conjugation of siRNA to Polymers and Knockdown Activity ofthe siRNA Polymeric Conjugates

ApoB siRNA sequences and PCR primers were prepared as described inWO/2010/054266. Preparation of thiolated siRNA was as follows. To a 15mL Falcon tube was added tris(2-carboxyethyl)phosphine hydrochloride(1.0 mg, 3.5 μmol, TCEP) followed by NaHCO₃ (1.2 mg, 14.0 μmol), H₂O(500 μL) and ApoB-SSC₆OH duplex (5.0 mg, Agilent Technologies). Thismixture was allowed to stand at room temperature. After 30 min, 5.0 MNaCl (20.0 μL) was added followed by cold (−20° C.) 100% EtOH (5.0 mL).The mixture was placed into a −80° C. freezer for 30 min to achievecomplete RNA precipitation. The Falcon tube was then centrifuged topellet the RNA. The mother liquor was removed and the remaining RNApellet was triturated using cold (+4° C.) 70% EtOH (1×1.0 mL). Theremaining RNA pellet was then dissolved in isotonic glucose (5.0 mL,5.05 wt % glucose, 10 mM HEPES, pH 7.4) or other suitable buffer to givean aqueous RNA solution with RNA concentration at 0.7 μg/μL (by UVanalysis).

Lyophilized polymer was dissolved in 100% EtOH to a stock concentrationof 100 mg/ml. The polymer was then slowly diluted into isotonic glucosewith 20 mM HEPES and gently mixed. Reduced siRNA was then added bringingeach component to the desired final concentration. Conjugation tookplace overnight at room temperature before dosing. A separateconjugation reaction was run for each dose group. The conjugationreactions were analyzed by gel electrophoresis (20% polyacrylamide,1×TBE gel from Invitrogen, 1×TBE buffer for ca. 1 h at 200 V, stained in50 mL 1×TBE with 2.5 μL SYBR gold for 15 min). Aliquots of the 3.0 mL invivo samples prepared above were withdrawn and a dilution series wasprepared. For example, the sample (4.0 μL) was diluted with blue-dyeloading buffer (6.0 μL) giving a sample with final RNA concentration of0.04 μg/μL. Then 4 μL of this diluted sample was applied to the gel.Similarly, the sample (4.0 μL) was treated with DTT (1.0 μL, 1.0 Msolution) for 10 minutes before being diluted further with 2.5% SDS (2.0μL) and loading buffer (3.0 μL) giving a sample with final RNAconcentration of 0.04 μg/μL. Then 4 μL of this reduced solution was alsoapplied to the gel for analysis. Conjugation efficiencies were greaterthan 90%.

The knockdown activity of the resulting conjugated siRNA formulationswas tested as described below.

Example 17. Knockdown Activity and Liver Toxicity siRNA PolymericConjugates

Female Balb/C mice, age 8 weeks at dosing, or CD-1 mice, age 7 weeks atdosing, were housed in groups of 4-5 animals. Food, water, temperatureand humidity are according to vivarium performance standards (SOPs)which are in accordance with the 1996 Guide for the Care and Use ofLaboratory Animals (NRC), AAALAC-International, and Seattle Children'sResearch Institute Institutional Animal Care and Use Committee (IACUC).Animals were acclimated to the facility for at least 5 days prior toexperimentation. A single dose of the formulated conjugate from Example9 or control of 10 mL/kg, 0.2 mL per 20 g mouse was administered i.v.via tail vein on Day 0. Study endpoints (24 hr or 48 hr) included:Clinical Observations; quantification of ApoB mRNA in liver; andquantification of aspartate transaminase (AST) and alanine transaminase(ALT) at necropsy.

Blood was collected immediately prior to necropsy via retro-orbitalsinus and placed into serum separator tube. Blood was processed to serumand samples were stored at 4° C. until sent to Phoenix CentralLaboratories (Seattle, Wash.) for analysis of liver transaminases.

After blood samples were collected, animals were euthanized using CO₂asphyxiation followed by cervical dislocation, and the abdomen wasopened. Approximately 200 mg of liver tissue was excised from the leftlateral lobe and placed in sterile 24-well cell culture platescontaining 2 mL of RNAlater (Applied Biosystems) solution. Usingscissors treated with RNaseZap (Ambion), the tissue was chopped intosmall pieces to allow penetration of RNAlater solution into tissue.Samples were stored at 4° C. until processed for total RNA isolation.

Dosing solutions were analyzed by dynamic light scattering (Malvern, UK)to determine particle size.

ApoB mRNA was measured using quantitative PCR by SYBR green chemistry asdescribed in Applied Biosystems tutorial Essentials of qPCR. In brief,approximately 50 mg liver tissue was transferred from RNAlater into TRIReagent in individual sterile homogenization tubes. The tissue was thenhomogenized and the RNA fraction was extracted. Total RNA was isolatedusing the MagMax-96 for Microarrays Total RNA Isolation Kit (AppliedBiosystems). RNA samples were diluted to 30 ng/ml for cDNA synthesis. 10μl of RNA was subjected to random primed reverse transcription using theHigh Capacity cDNA Reverse Transcription Kit (Applied Biosystems) anddiluted 1:5 in nuclease free water for use in the qPCR reactions.

For determining relative expression, RNA from each Vehicle sample waspooled into one sample for cDNA synthesis which is referred to as thePool sample. The Pool sample was run on each PCR plate to serve as thereference sample for relative expression. Primer sets specific to ApoBas well as two internal normalizing genes (Calnexin and HPRT) were runin triplicate for each sample on the same PCR plate. StepOne™ software(Applied Biosystems) was used to calculate the relative quantity of thetarget gene ApoB normalized to the two internal normalizer genes andthen relative to the POOL sample using the comparative C_(T) (ΔΔC_(T))method. Each Vehicle sample was assayed in addition to the Pool Sampleto show that the Pool Sample was representative of the individualVehicle control samples.

Quantification of serum ALT and AST levels was done at Phoenix CentralLaboratories (Everett, Wash.). Body weights were collected for eachanimal prior to dosing and prior to necropsy. The percent weight changewas then calculated. Descriptive statistics (average and standarddeviation [SD]) were determined for each group and dose level for ApoBrelative gene expression (RQ), ALT and AST levels, and body weight.

Results of the experiments are summarized in the Tables 6 and 7.

TABLE 6 Liver transaminases (AST and ALT), relative gene expression(RQ), and % knockdown for ApoB mRNA in the liver of Balb/c 24 Hoursafter administration of polymer-siRNA conjugates or vehicle; SD =standard deviation. Polymer SiRNA ALT Dose Amount AST (U/L) (U/L)Polymer (mg/kg) (mg/kg) Average AST (U/L) ± SD Average ALT (U/L) ± SD RQRQ ± SD % KD P1 150 7.5 386 328 242 248 0.0464 0.0154 95 125 6.3 151 43106 46 0.1008 0.0431 90 100 5.0 130 34 69 26 0.3064 0.0979 69 75 3.8 16371 51 7 0.5629 0.1092 44 50 2.5 114 54 33 23 0.9450 0.1470 5 25 1.3 10314 42 3 0.9441 0.1062 6 P2 150 7.4 165 74 93 40 0.0767 0.0453 92 125 6.2126 18 69 29 0.1683 0.1014 83 100 4.9 116 38 64 20 0.1634 0.0856 84 753.7 130 44 52 16 0.2438 0.1326 76 50 2.5 108 30 60 19 0.7364 0.0839 2625 1.2 92 13 42 4 0.8461 0.1782 15 P3 150 5.6 7398 7374 6734 5937 0.00340.0033 100 125 4.7 967 1067 1102 1357 0.0182 0.0127 98 100 3.8 244 137330 321 0.0382 0.0208 96 75 2.8 282 174 276 214 0.0336 0.0270 97 50 1.9104 29 58 11 0.1720 0.0630 83 25 0.9 98 44 38 5 0.7454 0.1703 25 P5 1504.8 50634 11596 47918 9205 0.1472 0.0536 85 125 4.0 42285 12158 3711811460 0.0212 0.0191 98 100 3.2 13574 10505 11712 8202 0.0047 0.0037 9975 2.4 772 279 1313 426 0.0254 0.0053 91 50 1.6 261 136 540 351 0.07750.0342 92 25 0.8 114 65 145 236 0.3482 0.0192 65 15 0.5 82 11 31 120.6133 0.0687 39 Vehicle n/a n/a 104 40 42 14 0.9645 0.1079 0

TABLE 7 Liver transaminases (AST and ALT), relative gene expression(RQ), and % knockdown (% KD) for ApoB mRNA in the liver of CD-1 mice at48 Hours after administration of polymer-siRNA conjugates or vehicle; SD= standard deviation. Polymer SiRNA ALT Dose Amount AST (U/L) AST (U/L)ALT Polymer (mg/kg) (mg/kg) Average (U/L) ± SD Average (U/L) ± SD RQ RQ± SD % KD P1 125 3.0 130 48 53 38 0.3047 0.2201 70 42 1.0 100 41 60 640.5208 0.1078 48 19 0.3 138 56 46 26 0.9544 0.1055 5 P4 150 3.6 128 4842 18 0.8884 0.2323 11 125 3.0 148 122 48 25 0.9012 0.0822 0 100 2.4 220104 81 59 0.8260 0.1177 17 75 1.8 111 55 39 20 1.1688 0.3112 0 42 1.0254 168 82 51 1.1727 0.2261 0 P5* 75 2.4 5641 10866 8022 15793 0.12580.0746 87 50 1.6 219 259 189 314 0.3962 0.1617 60 25 0.8 114 35 46 210.6324 0.1369 37 P6 125 5.0 156 95 146 129 0.0348 0.0050 97 75 2.45 8728 93 64 0.0444 0.0075 96 50 1.6 67 13 42 10 0.1599 0.0511 84 30 4.0 6410 32 10 0.2618 0.1397 74 20 2.45 196 202 47 12 0.4100 0.1579 59 10 1.679 32 35 16 0.7279 0.1815 27 Vehicle n/a n/a 72 17 33 4 1.0185 0.0616 0*Experiment stopped at 24 Hours due to observed toxicity.

Example 18. Formulation of Block Copolymers of the Invention and mRNAComplexes

A The polymers P7 of Example 9, P9 of Example 12 or P10 of Example 13were solubilized at 100 mg/mL in 200 proof ethanol and then diluted to20 mg/mL in 20 mM HEPES buffer at pH 7.4 containing 5% glucose (HEPESbuffer). The individual polymer stock solutions were kept at −20° C.until used. The P7 and P9 stock solutions were mixed together at a 77%and 23% molar ratio prior to use. P10 and P7 were also mixed togetherbut at a 30% and 70% molar ratio prior to use. Typically, for P9/P7 793μL of P9 at 20 mg/mL in HEPES buffer was added to 207 μL of P7 in bufferfor a final volume of 1 mL and a final polymer concentration of 20mg/mL. Typically, for P10/P7 365 μL of P10 at 20 mg/mL in HEPES bufferwas added to 635 μL of P7 in buffer for a final volume of 1 mL and afinal polymer concentration of 20 mg/mL. The FLuc (firefly luciferase)mRNA stock solution at 2 mg/mL in 10 mM Tris-HCL (pH7.5) from TriLinkBiotechnologies (San Diego, Calif., USA)(catalog number L-6107) wasdiluted to 0.2 mg/mL in HEPES buffer. The polymer mRNA formulation wasassembled at a N/P ratio of 20 by adding 1 mL of the diluted polymerstock solution at 20 mg/mL to 1 mL of mRNA at 0.2 mg/mL in HEPES bufferunder a mild vortex agitation. The formulation was kept at 4° C.overnight prior to in vivo dosing. The formulations were dosedintravenously at 1 mg/kg mRNA and 100 mg/kg of polymer.

The formulation particle size was measured by adding 10 μL offormulation to 90 μL of HEPES buffer into a disposable micro-cuvette andanalyzed using the Malvern Instrument ZETASIZER NANO-ZS. The formulationzeta-potential at pH 7.4 was measured by adding 10 μL of formulation to740 μL of HEPES buffer into a disposable 1 mL cuvette. The zeta dip cellwas inserted into the 1 mL cuvette and the formulation was analyzedusing the ZETASIZER NANO-ZS. The zeta-potential was also measured at pH4 as described above by adding 10 μL of formulation to 740 μL of 20 mMacetate buffer pH 4 containing 5% glucose. The ability of the polymerformulation to compact the mRNA was measured in a 96 well plate using aSYBR Gold dye accessibility assay. Typically, 50 μL of the polymerformulation at 0.01 mg/mL mRNA was added to 150 μL of diluted SYBR Goldstock solution (1 μL of Stock SYBR Gold in 3 mL of HEPES buffer) andincubated for 15 minutes at room temperature with agitation (100 RPM).The fluorescence was read at an excitation wavelength of 495 nm andemission wavelength of 538 nm. The percent dye accessibility wascalculated by dividing the fluorescence intensity of the formulated mRNAby the fluorescence intensity of the free mRNA×100.

Example 19. In Vivo Testing of Polymer-mRNA Formulations

Female CD-1 mice (6-8 weeks old) were used for in vivo testing ofpolymer-FLuc mRNA formulations. The formulations were dosedintravenously at 1 mg/kg of mRNA and 100 mg/kg of total polymer dose,with 3-5 mice injected per group. Mice injected with HEPES buffer aloneand HEPES buffer containing unformulated FLuc mRNA at 1 mg/kg were usedas controls. All mice were given a final volume of approximately 0.25 mlor 10 mL/kg based on individual body weights.

The in vivo expression of luciferase was evaluated by detectingluminescence in mice using the Xenogen IVIS Lumina II Imaging System(Caliper Life Sciences, now Perkin Elmer). The imaging was performed at3 and 6 hours following dosing. 15 minutes prior to imaging, each mousereceived 0.25 ml of D-luciferin (Perkin Elmer), a luciferase substrate,at 15 mg/ml (dissolved in 1×PBS) by intra-peritoneal injection. A fewminutes before imaging, mice were place in an isoflurane chamber toinduce anesthesia (isoflurane concentration at ˜3%). Subsequently, micewere moved into the IVIS imaging chamber, with the snout connected to anisoflurane-filled nose cone with the mouse's ventral side up. Theluminescence images were acquired using Living Image software (CaliperLife Sciences) with the exposure time, binning and F/Stop remain thesame throughout the study. Mice were put back to the cage as soon as theimaging was finished and they recovered within 1-3 minutes.

After the image acquisition was finished for all mice, the luminescenceresults were analyzed using Living Image software. Briefly, the colorscale of each image was first adjusted to display specific luminescencesignal and eliminate background signal. Then a region of interest (ROI)for the liver was defined using the ROI tools, and ROI measure buttonwas clicked to show the photon flux data. Total flux (photons/sec) ofthe ROI on each animal was used to represent the intensity ofluminescence. Total flux was averaged from all 5 mice for eachformulation group for comparison.

Table 8 displays luminescence values in the liver for animals treatedwith either P9/P7+FLuc mRNA, P10/P7+FLuc mRNA, buffer, or unformulatedmRNA. Data was acquired at 3 and 6 hours post dose. While neither ofbuffer or unformulated mRNA showed any activity, all mice receivingeither of two polymer-mRNA formulations demonstrated strong luminescencesignal in the liver.

TABLE 8 Luminescence results from individual animals at 3 and 6 hrs postdosing. 3 hr Luminescence 6 h Luminescence Total Flux Total FluxFormulation Animal ID (photons/sec) Ave SD (photons/sec) Ave SD Buffer 11.57E+05 1.28E+05 2.56E+04 1.25E+05 1.57E+05 3.04E+04 2 1.08E+051.60E+05 3 1.20E+05 1.86E+05 Unformulated 4 4.56E+04 1.11E+05 5.98E+041.77E+05 1.14E+05 7.43E+04 FLuc mRNA 5 1.63E+05 3.19E+04 6 1.23E+051.34E+05 PRX392/PRX367 + 7 9.17E+07 5.11E+07 2.48E+07 7.38E+07 3.59E+072.58E+07 FLuc mRNA 8 5.27E+07 2.85E+07 9 3.36E+07 3.42E+07 10 2.89E+072.22E+06 11 4.88E+07 4.09E+07 PRX398/PRX367 + 12 1.63E+08 7.18E+077.31E+07 1.02E+08 3.95E+07 4.32E+07 FLuc mRNA 13 1.68E+07 1.06E+07 142.00E+07 1.06E+07 15 1.92E+07 6.77E+06 16 1.40E+08 6.73E+07

Example 20. In Vivo Testing of Knockdown Activity and Liver Toxicity ofsiRNA Polymer Formulations of Beta-Catenin and MET siRNA in aHepatocellular Carcinoma (HCC) Mouse Model

The gene knockdown response induced by β-catenin and MET siRNAs in asynthetic/transgenic HCC mouse model was evaluated following singledoses of β-catenin, MET or a combination of β-catenin and MET siRNAs.

Female FVB mice (6-8 wk) received tail vein injections of humanbeta-catenin (ΔN90) & human MET DNA plasmids (10 μg each+0.8 μg of SBplasmid; 2 ml total volume) to induce HCC. At week 4, a single dose ofbeta-catenin siRNA (a dsRNA molecule where the sense strand isnucleotide sequence of SEQ ID NO:54 and the antisense region isnucleotide sequence of SEQ ID NO:78 which is also designated as si033),MET siRNA (a dsRNA molecule where the sense strand is nucleotidesequence of SEQ ID NO:1 and the antisense region is nucleotide sequenceof SEQ ID NO:27 which is also designated as si034), a combination(si033+si034), or ITG buffer was given at week 4. The polymer waspolymer P6. Animals were sacrificed 1, 2, 3, 4, 6, or 10 days aftersiRNA dosing. Liver/tumor tissue samples were collected for PCR andWestern analyses using standard techniques known in the art. Serumsamples were evaluated for serum chemistry including alanineaminotransferase (ALT).

Formulations for dosing were prepared by taking lyophilized polymer(polymer P6) and dissolving it in ITG to a stock concentration of 60mg/ml. The polymer is then diluted in ITG to 7.5 mg/ml with 0.3 mg/mlreduced thiolated siRNA (either si033 or si034). The conjugationreaction is incubated at room temperature overnight. Formulations werestored in glass vials under argon at 4° C. throughout dosing. si033 andsi034 were formulated with polymer P6 as described above. Animals weregiven IV injections of either 3.0 mg/kg formulated si033, 3.0 mg/kgformulated si034, or 1.5 mg/kg each of a combination of formulated si033and si034 as described in Table 9. All animals were given a final dosingvolume of approximately 0.25 ml or 10 mL/kg based on body weight.

TABLE 9 Study Details Polymer siRNA Sacrifice # Animals/ Group siRNAsiRNA ID Polymer (mg/kg) (mg/kg) # Doses Timepoints Group 1 β-cateninsi033 PRX231-6 75 3 1 24 hr 6 2 si033 75 3 1 48 hr 6 3 si033 75 3 1 72hr 6 4 si033 75 3 1 96 hr 6 5 si033 75 3 1 6 days 6 6 si033 75 3 1 10days 6 7 MET si034 PRX231-6 75 3 1 24 hr 6 8 si034 75 3 1 48 hr 6 9si034 75 3 1 96 hr 6 10 si034 75 3 1 6 days 6 11 si034 75 3 1 10 days 512 Combination of si033, si034 PRX231-6 75 1.5 each 1 48 hr 6 13β-catenin si033, si034 75 1.5 each 1 96 hr 6 14 and MET si033, si034 751.5 each 1 6 days 6 15 si033, si034 75 1.5 each 1 10 days 6 16 BufferNone None N/A N/A 1 72 hr 5 17 None N/A N/A 1 10 days 6

Human β-catenin mRNA was knocked down relative to human MET mRNA throughday 4 after β-catenin siRNA (si033)/polymer dosing as shown in FIG. 1.

Human MET mRNA was knocked down relative to β-catenin mRNA through day10 after MET siRNA (si034)/polymer dosing as shown in FIG. 2.

The combination treatment upon the administration of a combinationformulation of si033 and si034 with polymer P6 showed variable mRNA KDas shown in FIG. 3.

β-catenin and Met expressions levels relative to GAPDH followingβ-catenin siRNA (si0333)/polymer treatment are shown in FIGS. 4 and 5respectively

β-catenin and Met expressions levels relative to GAPDH following MetsiRNA (si034)/polymer P6 treatment are shown in FIGS. 6 and 7respectively.

No significant changes were seen in alanine aminotransferase (ALT)levels upon the administration of formulations of si033 with polymer P6,si034 with polymer P6 or a combination of si033 and si034 with polymerP6 as shown in FIGS. 8 and 9.

Example 21: Synthesis of Macro-CTAs

By a process similar to that described in Example 8.1, the followingchain transfer agents were prepared:

NAG-C5-PEG₃₆-ECT

NAG-C5-PEG₂₄-amido-PEG₂₄-ECT

2-Morpholinoethyl-amido-ECT

Boc-Aminoxy-PEG₁₁-ECT

Boc-Aminoxy-PEG₃-ECT.

Example 22. Synthesis of Polymer NAG-PEG0.6KDa-[PEGMA(4-5,100%)]3.45KDa-b-[DMAEMA(35.8%)-BMA(47.5%)-PAA(9.2%)-PDSMA(7.5%)]6.6KDa(P11)

The synthesis of polymer P11 was conducted in two polymerization steps,a first block (conjugation block) polymerization (see FIG. 13A) and asecond block (endosome release block) polymerization (see FIG. 13B).

First Block (Conjugation Block) Polymerization

TABLE 10 Reagent Table Amount Amount Reactant and MW Calc. ExperimentalProduct Lot Number (g/mol) Equiv. mmol (mg) (mg) NAG- MQ-03-12-2 1151.451 1.817708 2093 2093 PEG0.6KDa- ECT PEGMA4-5 MKBN1112V 300.0 15.528.17448 8452 8454.2 AIBN 102413 164.21 0.05 0.090885 14.92 c = 1.0703mg/g (recrystallized Target AIBN Oct. 24, 2013) soln = 13.9401 g, used13.9402 g DMF DX1727-7 73.09 n/a n/a 14380 14391.9

Polymer Synthesis

AIBN/DMF (13.9402 g of 1.0703 mg/g AIBN in DMF) was added to the CTA(2.093 g; 1.817708 mmol) in a 40 mL reaction vessel and mixed todissolve the CTA. DMF was then added until the total weight of DMF was14.3919 g. Then PEGMA4-5 (8454.2 mg, 28.18 mmol, filtered throughaluminum oxide [activated, basic, Brockmann I]) was added. This mixturewas vortexed for several minutes to give a homogeneous stock solutionand transferred to a 50 mL round-bottom flask. A T₀ sample (40 μL) waspulled and stored at −20° C. for monomer incorporation determination.The solution was then cooled to 0° C. using an ice bath. The solutionwas degassed by bubbling nitrogen into the solution for 47 min(maintained at 0° C.), followed by flushing the head space with nitrogenfor an additional 4 min (total nitrogen time of 51 min). The flask wasmoved to room temperature for 10 min and then placed in a pre-heated oilbath (stir speed was set at 350 rpm, internal temperature=65° C.(thermocouple)).

After 1 h 45 min, the reaction was stopped by introducing oxygen (threeneedles inserting into the rubber septa) followed by opening the cap andthen placing the flask in an ice bath. A T_(f) sample (40 μL) was pulledand stored at −20° C. for monomer incorporation determination.

Polymer Purification

The reaction solution was diluted with MeOH (˜60 mL), transferred todialysis membranes (Spectrum Labs, Spectrum Spectra/Por*6 DialysisMembrane Tubing MWCO: 2000) and dialyzed against MeOH (5×4000 mL) for 7days. Samples were taken for GPC, HPLC, and NMR analyses.

Analytical Testing:

NMR Analysis

A small aliquot of the dialysis solution (ca. 500-1000 μL) was withdrawnfrom the dialysis tubing and placed into a tared vial. The solution wasthen evaporated using a rotary evaporator. Once the solvents wereremoved the vial was transferred to a high vacuum line and placed underhigh vacuum for 48 h. Then the compound (24 mg) was dissolved in 800 μLmethanol-d4 and a proton NMR spectrum was collected.

The ¹H NMR of polymer P11 block 1 indicated a polymer was prepared byincorporating PEGMA (4-5). The ¹H NMR was consistent for proposedstructure.

Analytical GPC Analysis

Overview

Polymers were analyzed by gel permeation chromatography (GPC) inDMF/LiBr with a triple detection method using a Viscotek system (GPCmaxVE-2001). The GPC analysis used multiple detectors, including a ViscotekRI detector, S3210 UV/Vis, and 270 Dual Detector (light scattering). The270 Dual Detectors contains a differential viscometer detector, anadvanced low angle (7°) light scattering detector (LALS) and right anglelight scattering detector (RALS). OmniSEC software was used to calculatethe absolute molecular weight of the polymer.

Procedure

Sample Preparation

-   -   1. Polystyrene GPC Standard (Polystyrene, 20,000, analytical        standard for GPC, Aldrich (Fluka) #81407).    -   2. Dissolve the GPC standard polymer in degassed DMF/1% LiBr (˜3        mg/ml, record actual concentration).    -   3. Filter through a 0.45 μm nylon filter (Acrodisk 13 mm syringe        filter, Pall Life Sciences #4426T) into an autosampler vial.

Polymer samples

-   -   1. Dissolve polymer sample in degassed DMF/LiBr (˜8 mg/ml,        record actual concentration).    -   2. Filter through a 0.45 μm nylon filter (Acrodisk 13 mm syringe        filter, Pall Life Sciences #4426T) into an autosampler vial.        Columns and Settings

Columns and Parameters:

-   -   1. Guard Column: PolarGel-M, 50×7.5 mm (P/N: PL1117-1800)    -   2. Columns: 2×PolarGel-M, 300×7.5 mm (P/N: PL1117-6800)        (PolarGel-M GPC columns are packed with low swell, macro porous        copolymer beads that have a surface of balanced polarity,        comprising hydrophobic and hydrophilic components, Polymer Labs        (Agilent).    -   3. Eluent: DMF/1% LiBr (w/v), filtered through a 0.2 μm Nylon        Filter    -   4. Flow Rate: 0.7 mL/min    -   5. Injection Volume: 60, 80, 100 and 120 μL    -   6. Column Temperature: 50° C.    -   7. Viscotek detectors: S3210 UV/Vis, RI detector, and 270 Dual        Detector    -   8. Analysis run time=40 min.

Polymer Analysis

One injection of polystyrene (20 KDa, 100 μt, GPC standard polymer) isneeded for the polymer analysis. The GPC data is worked up by pickingbaseline and polymer peaks in the RI and RALS detector traces usingOmniSEC software. In the OmniSEC software, a new method is written basedon the Polystyrene (20 KDa) standard analysis:

-   -   1. Method—New—Blank—Multidetectors—Homopolymers    -   2. Choose Detectors: RI and RALS    -   3. Enter Standard Name: Polystyrene (20 KDa)    -   4. Enter Standard Name    -   5. RI for solvent=1.43    -   6. Save method    -   7. Calibrate method    -   8. Five injections of each PRX polymer are needed for the        polymer analysis (60, 80, 100, 120, and 140 μL).        -   a. Open traces for the five polymer injections in the            OmniSEC software        -   b. Fix baseline and peak pick for each sample        -   c. Close all files        -   d. Determine Polymer dn/dc            -   i. In the OmniSEC software, open trendview:                tools—trendview            -   ii. Set view to dndc            -   iii. Open method from step 4.3aV above            -   iv. Open polymer series: file—open            -   v. Calculate dn/dc            -   vi. Record polymer dn/dc value    -   9. Determine Polymer Molecular Weight and Polydispersity (PDI)        -   In the OmniSEC software, open the polymer trace (100 μL            injection)        -   In the method, enter the polymer dn/dc value from step            4.3bIIv above        -   Calculate the molecular weight (Σ)        -   Record values for Mn, Mw, and PDI. Reported values for PRX            polymers will be Mn values.

Analytical GPC Results

Mn=4,600 g/mol, PDI=1.12, dn/dc=0.05932

Monomer Incorporation by HPLC

The analysis of the HPLC results indicate the following monomerincorporation ratios in the polymer: NAG-PEG₁₂-[PEGMA₁₀₀%]_(3.45k). Theoverall conversion of this polymerization reaction was 23.4% with PEGMAincorporation at 100%.

TABLE 11 Monomer Incorporation Calculations Peak Peak Peak Area 1 Area 2Area 3 PEGMA 4-5 7761198 7911522 7867785 PDSMA 0 0 0 PEGMA 4-5 60328326040105 5957546 PDSMA 0 0 0 % conversion 22.27 23.65 24.28 PEGMA 4-5 %conversion BPAM % conversion PDSMA 0.00 0.00 0.00 mol PEGMA 4-5 inc.0.2227 0.2365 0.2428 mol BPAM inc. 0 0 0 mol PDSMA inc. 0 0 0 Overall %Conversion Std Dev Total mol in Polymer 0.2227 0.2365 0.2428 23.40 1.03Average Std Dev % PEGMA 4-5 (in 100.00 100.00 100.00 100.00 0.00polymer) % BPAM (in polymer) 0.00 0.00 0.00 0.00 0.00 % PDSMA (in 0.000.00 0.00 0.00 0.00 polymer) BPAM PDSMA PEGMA 4-5 Feed Feed Feed 1 0

Isolation of the Final Polymer

Once the final GPC analysis was determined, then the dialysis solutionwas transferred to a 40 mL reaction vial. The solvent was removed underreduced atmosphere followed by high vacuum (approx. 20 h) to afford1.980 g of polymer (yield 23.7%).

Second Block (Endosome Release Block) Polymerization

TABLE 12 Reagent Table Reactant Actual and FW Amt Amt Product Lot Number(g/mol) D Eq mmol (mg) (mg) Macro- DR-01-53 4600.00 1 0.430435 1980 1980CTA PAA SRG-255- 114.14 0.951 28.5 12.26739 1400.20 1419.3 169C DMAEMA11024JE 157.22 0.933 28.5 12.26739 1928.68 1924 BMA MKBL3019V 142.200.894 56 24.10435 3427.64 3437.7 PDMSA MQ-03-22- 255.4 6 2.582609 659.49663.3 vial 4 AIBN 102413 164.21 0.1 0.043043 7.07 c = 1.0910 mg/g TargetAIBN soln = 6.4801 g, used 6.4837 g DMF DX1727-7 73.09 0.944 n/a n/a14060 14148.6

Monomer Analytical Analysis

PDSMA was analyzed by GPC immediately prior to use to confirm that nohigh molecular weight polymer was present as a result ofhomopolymerization.

Polymer Synthesis

AIBN/DMF solution (6.4837 g/g; 1.0910 mg/g AIBN in DMF) was added to themacro-CTA (polymer P11 block 1, 1.980 g) in a 40 mL reaction vessel. DMFwas then added until the total weight of DMF was 14.1486 g and thesample was mixed to dissolve the macro-CTA. BMA (3437.7 mg, 24.10435mmol, filtered through Aluminum oxide [activated, basic, Brockmann I]),PAA (1419.3 mg, 12.2674 mmol, monomer not purified, 2-propylacrylicacid, lot # SRG-255-169C), DMAEMA (1924 mg, 12.2674 mmol, filteredthrough Aluminum oxide [activated, basic, Brockmann I], and PDSMA (663.3mg, 2.5826 mmol, batch MQ-03-22-vial 4) were added to the reactionsolution. The mixture was vortexed for several minutes to give ahomogeneous stock solution and transferred to a 50 mL round-bottomflask. A T₀ sample (40 μL) was pulled and stored at −20° C. for monomerincorporation determination. The solution was then cooled to 0° C. usingan ice bath. The solution was degassed by bubbling nitrogen into thesolution for 46 min (maintained at 0° C.), followed by flushing the headspace with nitrogen for an additional 5 min (total nitrogen time of 51min). Then the flask was sealed with parafilm and placed into apre-heated oil bath (stirring speed was 350 rpm, internaltemperature=59° C. (thermocouple)).

After 8 h, the reaction was stopped by introducing oxygen (three needlesinserting into the rubber septa) followed by opening the cap and thenplacing the flask in an ice bath. A T_(f) sample (40 μL) was pulled andstored at −20° C. for monomer incorporation determination.

The reaction was then diluted with approximately 35 mL of acetone andprecipitated into a stirred mixture of ether/hexanes (1:3 v/v) in 50 mLcentrifuge tubes (10) first and then again into a large beaker with 900mL ether/hexanes (1:3 v/v).

Polymer Purification

The polymer dissolved with MeOH (100 mL), transferred to four individualdialysis membranes (Spectrum Labs, Spectrum Spectra/Por*6 DialysisMembrane Tubing MWCO: 2,000) and dialyzed against methanol (4×8000 mL;two 4 liter beakers) for 5 days. After the dialysis against methanol, itwas dialyzed against nanopure water using the same membrane (×7 over 5h). When the dialysis was complete, the solution was transferred to 10individual 20 mL tared vials, frozen (liquid nitrogen followed by driedice), and lyophilized for 6 days to afford 3.77 g of the final product(yield=78.2%). The final product was analyzed by UVNis (for PDS contentof the polymer, in DMF+TCEP), NMR (in methanol-d4), and GPC (DMF+LiBr).The final product was stored in glass vials with rubber septum that werepurged with argon and sealed with parafilm. The vials were stored at−20° C.

Analytical Testing

¹H NMR of Polymer P11 Block 1—Block 2 CD₃OD

NMR results were consistent with proposed structure. There was noevidence of remaining vinyl monomers as indicated by the lack of signalsbetween 5.4 and 6.5 ppm.

Analytical GPC

Results: Mn=11,200 g/mol (100 μL injection), PDI=1.57, dn/dc=0.0624

Monomer Incorporation by HPLC

Analysis of the monomer incorporation by HPLC results indicated theaverage overall conversion of this polymerization reaction was 30.24%with DMAEMA incorporation at 35.8%, PAA incorporation at 9.2%, BMAincorporation at 47.5%, and PDSMA incorporation at 7.5%. The analysis ofthe HPLC indicated the following monomer incorporation ratios in thepolymer:NAG-PEG₁₂-[PEGMA₁₀₀%]_(3.45k)-[BMA_(47.5%)-PAA_(9.2%)-DMAEMA_(35.8%)-PDSMA_(7.5%)]_(6.6k).

TABLE 13 Monomer Incorporation Calculations Enter monomer feed ratiosDMAEMA PDSMA PEGMA BMA Feed Feed Feed PAA Feed Feed 0.24 0.05 0 0.240.47 0.24 0.05 0 0.24 0.47 Enter peak areas for each monomer; 3injections required-- injection 4 may be left blank Peak Areas To inj 4To inj 2 To inj 3 To inj 1 DMAEMA 3242224 3277249 3047662 PDSMA 12868891288943 1258209 PAA 3245906 3256478 3189947 BMA 6297454 6256387 6229199Tf inj 4 Tf inj 2 Tf inj 3 Tf inj 1 DMAEMA 1684539 1764750 1793265 PDSMA684450 706950 711990 PAA 2778065 2915338 2867467 BMA 4215662 44405914387927 D % 48.04 46.15 41.16 conversion PDS % 46.81 45.15 43.41 P %14.41 10.48 10.11 conversion B % 33.06 29.02 29.56 conversion mol D inc.0.1153 0.1108 0.0988 mol PDS inc 0.0234 0.0226 0.0217 Overall %Conversion Std Dev mol P inc. 0.0346 0.0251 0.0243 mol B inc. 0.15540.1364 0.1389 Total mol 0.3287 0.2949 0.2837 30.24 2.34 inc. Average StdDev % D (in 35.1 37.6 34.8 35.8 1.5 polymer) % PDS (in 7.1 7.7 7.7 7.50.3 Poly) % P (in 10.5 8.5 8.6 9.2 1.1 polymer) % B (in 47.3 46.3 49.047.5 1.4 polymer)

Polymer PDS Content

Background Information:

Polymer molecular weight was calculated by analytical GPC. Monomerincorporation was determined by analytical HPLC and was used todetermine the theoretical amount of PDS groups incorporated into thepolymer during polymer synthesis (PDS/polymer chain). Actual PDS contentwas determined by UV/Vis spectroscopy following disulfide reduction andliberation of pyridine-2-thione.

The molar absorbtivity of pyridine-2-thione was determined to be ∈=5,695M⁻¹cm⁻¹ in DMF with λ_(max)=370 nm. At λ=370 nm, there was nearlynegligible absorption from the CTA or polymer.

Procedure

PRX polymer stock solution at 5-8 mg/mL in DMF was prepared. Actualconcentration was recorded. To 200 μL of polymer solution in aneppendorf tube, 6 μL of 0.5 M TCEP solution (Sigma #646547) was added.Following about a 5 min reduction, the solution was spun for about 1 minat max RPM to pellet precipitate.

Absorption was read on a Nanodrop ND-1000 spectrophotometer (λ=370 nm,path length=1 mm).

Analysis

The amount of pyridyl-2-thione (mol/1) was determined according to thefollowing formula: pyridyl-2-thione (mol/1)=(Abs 370/569.5 M⁻¹mm⁻¹).

The polymer concentration used in the assay was calculated according tothe following formula: Polymer analysis concentration(mg/ml)=(concentration of PRX polymer stock solution*0.2 ml)/0.206 ml.

The expected amount of pyridyl-2-thione (mol/1) was determined accordingto the following formula: Theoretical pyridyl-2-thione (mol/1)=Polymeranalysis concentration (mg/ml)/PRX Polymer Mn (g/mol)*TheoreticalPDS/polymer chain.

% of PDS groups found=[determined pyridyl-2-thione (mol/1)/Theoreticalpyridyl-2-thione (mol/1)]*100.

Actual PDS/polymer chain=determined pyridyl-2-thione (mol/1)/Theoreticalpyridyl-2-thione (mol/1)]*Theoretical PDS/polymer chain.

Results of analysis of the PDS content of the polymer indicated 2.28 PDS(62% of theoretical) groups per chain.

Conclusions

Polymer P11 was synthesized and released with the followingspecifications:NAG-PEG₁₂-[PEGMA_(100%)]_(3.45k)-[BMA_(47.5%)-PAA_(9.2%)-DMAEMA_(35.8%)-PDSMA_(7.5%)]_(6.6k).

By similar methods, the following polymers were synthesized according tothe following conditions shown in Tables 14-21, below.

-   -   a. Polymer P12:        NAG-PEG₃₆-[PEGMA300,100%]_(3.5k)-b-[BMA_(50%)-PAA_(9%)-DMAEMA_(35%)-PDSMA_(6%)]_(4.9k)

TABLE 14 P12 Block 1 Block 2 [M/CTA/I] [15.5/1/0.05] [120.5/1/0.1][concentration] 0.95M 2.3M Time 2 h 50 m 8 h 35 m Internal temp 65-66°C. 58-59° C. CTA = NAG-C₅-PEG₃₆-ECT; I = AIBN

-   -   b. Polymer P13:        NAG-PEG₂₄-amido-PEG₂₄-[PEGMA300,100%]_(3.6k)-b-[BMA_(50%)-PAA_(11%)-DMAEMA_(32%)-PDSMA_(7%)]_(3.8k)

TABLE 15 P13 Block 1 Block 2 [M/CTA/I] [15.5/1/0.05] [123/1/0.1][concentration] 0.68M 2.84M Time 3 h 45 m 10 h 15 m Internal temp 65-66°C. 58-59° C. CTA = NAG-C₅-PEG₂₄-amido-PEG₂₄-ECT; I = AIBN

-   -   c. Polymer P14: NAG-PEG₁₂-[PEGMA500        (100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(50%)-PAA_(8%)-PDSMA_(6%)]_(5.2k)

TABLE 16 P14 Block 1 Block 2 [M/CTA/I] [15.5/1/0.05] [123.9/1/0.1][concentration] 1.0M 2.36M Time 2 h 10 h Internal temp 65-66° C. 58-59°C. CTA = NAG-C₅-PEG₁₂-ECT; I = AIBN

-   -   d. Polymer P15: BocNO-PEG₁₁[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k)

TABLE 17 P15 Block 1 Block 2 [M/CTA/I] [16/1/0.05] [120/1/0.1][concentration] 1.16M 2.39M Time 1 h 45 m 5 h 25 m Internal temp 65-66°C. 58-59° C. CTA = Boc-Aminoxy-PEG₁₁-ECT; I = AIBN

-   -   e. Polymer P16: BOCNO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(33%)-BMA_(46%)-PAA_(14%)-PDSMA_(7%)]4.8k

TABLE 18 P16 Block 1 Block 2 [M/CTA/I] [16/1/0.05] [119.7/1/0.1][concentration] 1.16M 2.33M Time 1 h 45 m 7 h Internal temp 65-66° C.58-59° C. CTA = Boc-Aminoxy-PEG₁₁-ECT; I = AIBN

-   -   f. Polymer P17: BOCNO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k)

TABLE 19 P17 Block 1 Block 2 [M/CTA/I] [15.5/1/0.05] [119.6/1/0.1][concentration] 1.01M 2.58M Time 2 h 5 m 10 h Internal temp 65-66° C.58-59° C. CTA = Boc-Aminoxy-PEG₁₁-ECT; I = AIBN

-   -   g. Polymer P18: ECT-[PEGMA (300,        58%)-TFPMA_(42%)]_(5.14k)-b-[DMAEMA_(31%)-BMA_(49%)-PAA_(12%)-PDSMA_(8%)]_(5.03k)

TABLE 20 P18 Block 1 Block 2 [M/CTA/I] [29.3/1/0.05] [121/1/0.1][concentration] 1.6M 2.31M Time 2 h 15 m 8 h 15 m Internal temp 65° C.58-59° C. CTA = ECT; I = AIBN

-   -   h. Polymer P19: NAG-PEG₁₂-[PEGMA (300,        73%)-TFPMA_(27%)]_(4.55k)-b-[DMAEMA_(36%)-BMA_(46%)-PAA_(10%)-PDSMA_(7%)]_(5.33k)

TABLE 21 P19 Block 1 Block 2 [M/CTA/I] [20/1/0.05] [121/1/0.1][concentration] 0.97M 2.45M Time 2 h 5 m 9 h Internal temp 65° C. 58-59°C. CTA = NAG-C₅-PEG₁₂-ECT; I = AIBN

-   -   g. Polymer P30: BOCNO-PEG₁₁-[PEGMA (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k)

TABLE 22 P30 Block 1 Block 2 [M/CTA/I]^(c) [16/1/0.05] [120.5/1/0.1][concentration] 31-wt % 2.57M monomer in solvent Time 4 h 10 h 15 mInternal temp 65-66° C. 58-59° C. (approx.) ^(c)M = PEGMA 1000; CTA =BOC-Aminoxy-PEG₁₁-ECT; I = AIBN

-   -   h. Polymer P51: NAG-PEG₃₆-[PEGMA (500,        100%)]_(6.19k)-b-[PMAEMA_(31.6%)-BMA_(48.4%)-PAA_(13.1%)-PDSMA_(6.8%)]_(4.3k)

TABLE 23 P51 Block 1 Block 2 [M/CTA/I]^(c) [15.5/1/0.05] [121/1/0.1][concentration] 0.95M 2.38M Time 3 h 45 m 8 h Internal temp 65-66° C.58-59° C. (approx.) ^(c)M = PEGMA 500; CTA = NAG-C₅-PEG₃₆-ECT; I = AIBN

-   -   i. Polymer P52: NAG-PEG₃₆-[PEGMA (500,        100%)]_(6.19k)-b-[PMAEMA_(30.8%)-BMA_(50.8%)-PAA_(11.6%)-PDSMA_(6.8%)]_(3.5k)

TABLE 24 P52 Block 1 Block 2 [M/CTA/I]^(c) [15.5/1/0.05] [121/1/0.1][concentration] 0.95M 2.34M Time 3 h 45 m 4 h 50 m Internal temp 65-66°C. 58-59° C. (approx.) ^(c)M = PEGMA 500; CTA = NAG-C₅-PEG₃₆-ECT; I =AIBN

-   -   j. Polymer P53: NAG-PEG₄₈-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(31.4%)-BMA_(49.3%)-PAA_(9%)-PDSMA_(9%)]_(6.3k)

TABLE 25 P53 Block 1 Block 2 [M/CTA/I]^(c) [15.5/1/0.05] [108.4/1/0.1][concentration] 0.86M 2.32M Time 3 h 50 m 15 h 30 m Internal temp 65-66°C. 58-59° C. (approx.) ^(c)M = PEGMA 300; CTA = NAG-C₅-PEG₄₈-ECT; I =AIBN

-   -   k. By similar process, the following tri-NAG polymers were        prepared. Monomer % listed is the % monomer in the        polymerization reaction        -   i. Polymer P54: Tri-NAG-PEG₁₂-[PEGMA(300,            80%)-PDSMA_(10%)-BPAM_(10%)]_(6.4k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(4.2k).        -   ii. Polymer P55: Tri-NAG-PEG₁₂-[PEGMA(300,            80%)-PDSMA_(10%)-BPAM_(10%)]_(6.4k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(3.2k).        -   iii. Polymer P56: Tri-NAG-PEG₁₂-[PEGMA(300,            80%)-PDSMA_(10%)-BPAM_(10%)]_(6.1k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(4.9k).        -   iv. Polymer P57: Tri-NAG-PEG₁₂-[PEGMA(300,            80%)-PDSMA_(10%)-BPAM_(10%)]_(7k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(27.8k).

Example 23. Procedure for Synthesis of Polymer-Cationic PeptideConjugates

In the reaction vial, the polymer was dissolved in solvent (MeOH or DMF)at a concentration of 10 mM polymeric PDS, to which a stir bar wasadded. The resulting solution was stirred at moderate speed on a stirplate. Concurrently, peptide was dissolved in solvent (MeOH or DMF) atan approximate concentration of 10 mM. The concentration of peptidethiol was then determined by Ellman's assay. Using the thiolconcentration determined from Ellman's assay, the peptide stock solutionwas then adjusted to twice the reaction concentration by addingadditional solvent. The conjugation reaction was then conducted byslowly (˜2.5 mL/min) adding an appropriate volume (to afford the desiredamount of peptide/polymer) of the peptide stock solution to the polymersolution in the reaction vessel, while stirring. After the entire amountof peptide had been added, the reaction was allowed to proceed until thepeptide had been consumed. Progress of the conjugation was monitored byHPLC for release of pyridal-2-thione and consumption of peptide.

The HPLC assay for release of pyridal-2-thione and consumption ofpeptide was as follows: a small aliquot (60 uL) of the reaction solutionwas diluted (H2O+0.1% TFA) so the peptide concentration was 0.7 mM. Thediluted solution was then split into two equal volumes. Into one of thevolumes a 10% volume of 0.5 M aqueous TCEP was added, and mixed to fullyrelease pyridal-2-thione. Both aliquots were then applied to the HPLCwith UV monitoring at 210 nm and 370 nm. Loss of UV signal in elutionpeak corresponding to free peptide in the untreated reaction solutionwas used to indicate reaction progression. Comparison of the UV signalcorresponding to pyridal-2-thione between the untreated and TCEP treatedsamples was used to determine the effective conjugation of the peptideto the polymeric precursor. Completion of conjugation (consumptionof >95% of the added peptide) was usually reached in less than 1 hr.

After the conjugation reaction had reached completion, dipyridaldisulfide (1 eq to the amount of PDS groups on the original polymer) wasadded and the solution and incubated an additional 30 minutes. Thereaction solution was then diluted 2 fold with acetone and the conjugatewas precipitated with hexanes/Ether (3:1). The precipitate was collectedby centrifugation and decanted. The pellet was then dissolved in acetoneand the conjugate was washed by precipitation an additional two times.After the final precipitation, the isolated pellet was placed under highvacuum for 1 hour to further remove residual solvents. The driedconjugate was then dissolved in H₂0 and Lyophilized. Followinglyophilization, the product was weighed to determine the yield and asmall fraction (˜5 mg) was taken and dissolved in MeOH (100 mg/mL) thendiluted with H₂O+0.1% TFA to approximately 1 mg/mL of total peptide. Theresulting solution was analyzed by HPLC to analyze for any peptidespecies.

Analysis of the final material includes the % peptide loading on thepolymer, the amount of peptide/polymer chain, formation of peptide sideproducts (peptide dimer), and final amount of unconjugated peptide inthe final lyophilized conjugate.

According to the above procedure the following polymer-peptideconjugates were prepared:

-   -   Polymer P20 is the CK₁₀NH₂ conjugate of NAG-PEG₁₂-[PEGMA (300,        100%)]_(3.45k)-b-[BMA_(47.5%)-PAA_(9.2%)-DMAEMA_(35.8%)-PDSMA_(7.5%)]_(6.6k)—at        0.80 peptides/polymer;    -   Polymer P21 is the CK₁₀NH₂ conjugate of: NAG-PEG₁₂-[PEGMA500        (100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(50%)-PAA_(8%)-PDSMA_(6%)]_(5.2k)—at        1.06 peptides/polymer;    -   Polymer P22 is the CK₁₀NH₂ conjugate of NAG-PEG₃₆-[PEGMA300,        100%]_(3.5k)-b-[BMA_(50%)-PAA_(9%)-DMAEMA_(35%)-PDSMA_(6%)]_(4.9k)—at        0.96 peptides/polymer;    -   Polymer P23 is the CK₁₀NH₂ conjugate of        NAG-PEG₂₄-amido-PEG₂₄-[PEGMA300,        100%]_(3.6k)-b-[BMA_(50%)-PAA_(11%)-DMAEMA_(32%)-PDSMA_(7%)]_(3.8k)—at        0.94 peptides/polymer;    -   Polymer P24 is the CK₁₀NH₂ conjugate of        NAG-C5-PEG₂₄-amido-PEG₂₄-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k)—at        0.69 peptides/polymer;    -   Polymer P25 is the CK₁₀NH₂ conjugate of        NAG-C5-PEG5k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA-_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k)—at        0.75 peptides/polymer;    -   Polymer P26 is the CK₁₀NH₂ conjugate of ECT-[PEGMA (300,        58%)-NAG-C5-PEG₃₆        (42%)]_(19.9k)-b-[DMAEMA_(31%)-BMA_(49%)-PAA_(12%)-PDSMA_(8%)]_(5.03k)—at        0.48 peptides/polymer;    -   Polymer P27 is the CK₁₀NH₂ conjugate of NAG-PEG₁₂-[PEGMA (300,        73%)-NAG-C5-PEG₃₆        (18%)-TFPMA_(5%)]_(11k)-b-[DMAEMA_(36%)-BMA_(46%)-PAA_(10%)-PDSMA_(7%)]_(5.33k)—at        0.78 peptides/polymer;    -   Polymer P31 is the CK₁₀NH₂ conjugate of        NAG-C5-PEG10k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k)—at        0.99 peptides/polymer;    -   Polymer P32 is the CK₁₀NH₂ conjugate of        NAG-C5-PEG20k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (300,        100%)]_(3.8k)-b-[DMAEMA_(32%)-BMA_(47%)-PAA_(14%)-PDSMA_(7%)]_(4.0k)—at        1.25 peptides/polymer;    -   Polymer P33 is the CK₁₀NH₂ conjugate of        NAG-C5-PEG24-amido-PEG₂₄-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k)—at        1.29 peptides/polymer;    -   Polymer P34 is the CK₁₀NH₂ conjugate of        NAG-C5-PEG5k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k)—at        0.82 peptides/polymer;    -   Polymer P35 is the CK₁₀NH₂ conjugate of        NAG-C5-PEG10k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k)—at        0.97 peptides/polymer;    -   Polymer P36 is the CK₁₀NH₂ conjugate of        NAG-C5-PEG20k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(48%)-PAA_(9%)-PDSMA_(8%)]_(5.3k)—at        1.5 peptides/polymer;    -   Polymer P37 is the CK₁₀NH₂ conjugate of        NAG-C5-PEG24-amido-PEG₂₄-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA        (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k)—at        1.06 peptides/polymer;    -   Polymer P38 is the CK₁₀NH₂ conjugate of        NAG-C5-PEG5k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k)—at        1.12 peptides/polymer;    -   Polymer P39 is the CK₁₀NH₂ conjugate of        NAG-C5-PEG10k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k)—at        0.88 peptides/polymer;    -   Polymer P40 is the CK₁₀NH₂ conjugate of        NAG-C5-PEG20k-Ph-aldehyde(oxime)NO-PEG₁₁-[PEGMA (1000,        100%)]_(9.1k)-[DMAEMA_(32.3%)-BMA_(48.4%)-PAA_(11.8%)-PDSMA_(7.5%)]_(8.15k)—at        0.98 peptides/polymer;    -   Polymer P58 is the CK₁₀NH₂ conjugate of NAG-PEG₃₆-[PEGMA (500,        100%)]_(6.19k)-b-[DMAEMA_(31.6%)-BMA_(48.4%)-PAA_(13.1%)-PDSMA_(6.8%)]_(4.3k)—at        0.95 peptides/polymer;    -   Polymer P59 is the CK₁₀NH₂ conjugate of NAG-PEG₃₆-[PEGMA (500,        100%)]_(6.19k)-b-[DMAEMA_(30.8%)-BMA_(50.8%)-PAA_(11.6%)-PDSMA_(6.8%)]_(3.5k)—at        0.87 peptides/polymer;    -   Polymer P60 is the CR₁₀NH₂ conjugate of NAG-PEG₄₈-[PEGMA        (300,100%)]_(3.8k)-b-[BMA_(49.3%)-PAA_(9%)-DMAEMA_(31.4%)-PDSMA_(9%)]_(6.3k)—at        0.91 peptides/polymer;    -   Polymer P61 is the CR₁₀NH₂ conjugate of NAG-PEG₁₂-[PEGMA(500,        100%)]_(5.8k)-b-[DMAEMA_(35%)-BMA_(50%)-PAA_(8%)-PDSMA_(6%)]_(5.2k)—at        0.97 peptides/polymer;    -   Polymer P62 is the CR₁₀NH₂ conjugate of NAG-PEG₃₆-[PEGMA300,        100%]_(3.5k)-b-[BMA_(50%)-PAA_(9%)-DMAEMA_(35%)-PDSMA_(6%)]_(4.9k)—at        0.94 peptides/polymer;    -   Polymer P63 is the CK₁₀NH₂ conjugate of        Tri-NAG-PEG₁₂-[PEGMA(300,        80%)-PDSMA_(10%)-BPAM_(10%)]_(7k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(27.8k)—at        0.64 peptides/polymer (monomer % listed is the % monomer in the        polymerization reaction);    -   Polymer P64 is the CK₁₀NH₂ conjugate of        Tri-NAG-PEG₁₂-[PEGMA(300,        80%)-PDSMA_(10%)-BPAM_(10%)]_(6.1k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(4.9k)—at        0.85 peptides/polymer (monomer % listed is the % monomer in the        polymerization reaction);    -   Polymer P65 is the CK₁₀NH₂ conjugate of        Tri-NAG-PEG₁₂-[PEGMA(300,        80%)-PDSMA_(10%)-BPAM_(10%)]_(6.4k)-[BMA_(50%)-PAA₂₅%-DMAEMA_(25%)]_(3.2k)—at        0.70 peptides/polymer (monomer % listed is the % monomer in the        polymerization reaction);    -   Polymer P66 is the CK₁₀NH₂ conjugate of        Tri-NAG-PEG₁₂-[PEGMA(300,        80%)-PDSMA_(10%)-BPAM_(10%)]_(6.4k)-[BMA_(50%)-PAA_(25%)-DMAEMA_(25%)]_(42k)—at        1.1 peptides/polymer (monomer % listed is the % monomer in the        polymerization reaction).

Example 24. Preparation for the Oxime Ligation on Polymers

The boc-protected hydroxyl amine polymer (0.19 mmol) was dissolved in anexcess of neat TFA (4 mL) and stirred for one hour to deprotect thehydroxyl amine. The TFA was then removed from the polymer under reducedatmosphere (by RotoVap, 30 min). The resulting polymer was used withoutfurther purification. The oxime ligation reaction was started bydissolving 2.5 equivalents of the NAG-PEGx-Ph-aldehyde (0.5 mmol) in aminimum of DMSO (˜2 mL), and the resulting solution was then added tothe hydroxyl amine polymer. A small aliquot was taken for GPC and HPLCanalysis to determine the initial NAG-PEGx-PH-aldehyde content in thereaction. The ligation reaction was allowed to proceed for 16 hours.After 16 hours an aliquot of the reaction mixture was taken for finalGPC and HPLC determination of NAG-PEGx-content. The reaction was thenprecipitated in cold Hexanes/Ether (2:1) to recover the oxime ligatedpolymer. The precipitate was collected by centrifugation, decantingsolvent from the pellet. The pellet was then washed an additional twotimes by dissolving in acetone and precipitating in cold Hexanes/Ether.The final pellet was placed on high Vac overnight to remove residualsolvent.

According to the above procedure the following polymers were prepared:

-   -   Polymer P28 is the oxime ligated polymer between        NAG-C5-PEG24-amido-PEG24-PH-aldehyde and polymer P15;    -   Polymer P29 is the oxime ligated polymer between        NAG-C5-PEG5k-PH-aldehyde and polymer P15;    -   Polymer P41 is the oxime ligated polymer between        NAG-C5-PEG10k-Ph-aldehyde and polymer P15;    -   Polymer P42 is the oxime ligated polymer between        NAG-C5-PEG20k-Ph-aldehyde and polymer P15;    -   Polymer P43 is the oxime ligated polymer between        NAG-C5-PEG24-amido-PEG24-Ph-aldehyde and polymer P30;    -   Polymer P44 is the oxime ligated polymer between        NAG-C5-PEG5k-Ph-aldehyde and polymer P30;    -   Polymer P45 is the oxime ligated polymer between        NAG-C5-PEG10k-Ph-aldehyde and polymer P30;    -   Polymer P46 is the oxime ligated polymer between        NAG-C5-PEG20k-Ph-aldehyde and polymer P30;    -   Polymer P47 is the oxime ligated polymer between        NAG-C5-PEG24-amido-PEG24-Ph-aldehyde and polymer P17;    -   Polymer P48 is the oxime ligated polymer between        NAG-C5-PEG5k-Ph-aldehyde and polymer P17;    -   Polymer P49 is the oxime ligated polymer between        NAG-C5-PEG10k-Ph-aldehyde and polymer P17;    -   Polymer P50 is the oxime ligated polymer between        NAG-C5-PEG20k-Ph-aldehyde and polymer P17.

Example 25: Formulation of Polymers with FLuc mRNA

Polymers are solubilized in 300 mM sucrose/20 mM phosphate buffer, pH7.4 (SUP) plus 1% Tween 80 with agitation at 20 mg/mL for 1 hour. FLuc(firefly luciferase) mRNA stock solution at 1 mg/mL in 10 mM Tris-HCL(pH7.5) from TriLink Biotechnologies is diluted to 0.2 mg/mL in SUPbuffer. Using a microfluidics device, the polymer and mRNA solutions aremixed at a 1:1 volume, at 12 mL/minute, and at an N/P ratio typicallyaround 20. The final concentrations of the polymer and mRNA aretypically at 10 mg/mL and 0.1 mg/mL respectively in 0.5% Tween 80/SUPbuffer. The formulations are incubated at room temperature forapproximately 60 minutes prior to injecting mice.

The formulation particle size is measured by adding 10 μl of formulationto 90 μL of SUP buffer into a disposable micro-cuvette and analyzedusing the Malvern Instrument ZETASIZER NANO-ZS. The formulationzeta-potential at pH 7.4 is measured by adding 10 μl of formulation to740 μL of SUP buffer into a disposable 1 mL cuvette. The zeta dip cellis inserted into the 1 mL cuvette and the formulation is analyzed usingthe ZETASIZER NANO-ZS. The zeta-potential is also measured at pH 4 asdescribed above by adding 10 μl of formulation to 740 μL of 20 mMacetate buffer pH 4 containing 5% glucose. The ability of the polymerformulation to compact the mRNA is measured in a 96 well plate using aSYBR Gold dye accessibility assay. Typically, 50 μL of the polymerformulation at 0.01 mg/mL mRNA is added to 150 μL of diluted SYBR Goldstock solution (1 μL of Stock SYBR Gold in 3 mL of SUP buffer) andincubated for 15 minutes at room temperature with agitation. Thefluorescence is read at an excitation wavelength of 495 nm and emissionwavelength of 538 nm. The percent dye accessibility is calculated bydividing the fluorescence intensity of the formulated mRNA by thefluorescence intensity of the free mRNA×100.

Example 26: In Vivo Testing of Polymer-mRNA Formulations

FLuc mRNA was formulated with polymers P20, P22, or P23 as described inExample 25.

Female CD-1 mice (7-10 weeks old) were used for in vivo testing of thepolymer-FLuc mRNA formulations. The formulations were dosedintravenously at 1 mg/kg of mRNA and 100 mg/kg of total polymer dose,with 5 mice injected per group. Mice injected with vehicle only (SUPbuffer) wa used as a control. All mice were given a final dose volume ofapproximately 0.25 ml or 10 mL/kg based on individual body weights.

In vivo luminescence on live mice was detected using an IVIS Lumina IIImaging System (PerkinElmer, Hopkinton, Mass.) in connection with theLiving Image Software (version 4.3, PerkinElmer). Each mouse wasinjected with 250 μL of D-luciferin potassium salt (PerkinElmer, 15mg/mL, dissolved in PBS without magnesium and calcium)intra-peritoneally 10 minutes prior to imaging. Mice were sedated with2% isoflurane gas anesthesia right before imaging and subsequentlyplaced in the imaging chamber. The image acquisition was operated usingthe luminescent option in Living Image Software with the exposure timeset at auto or desired length (e.g., 20 seconds). The images wereanalyzed in Living Image Software using Region of Interest (ROI) tool toquantify the luminescence of each animal, which was expressed as totalflux (photons/second).

FLuc mRNA was quantified in liver tissue and blood using a quantitativePCR assay. Mice were sedated at the designated time points with 2%isoflurane gas anesthesia and then 200 μL of blood is collectedretro-orbitally. Whole blood was immediately diluted into 1 ml of TRIzolReagent (Life Technologies) mixed well and then placed on ice. Followinganimal sacrifice, 50-100 mg of liver tissue was placed in a sterile tubeand flash frozen in liquid nitrogen. Frozen liver samples were taken upin a sufficient volume of TRIzol Reagent to 100 mg/mL based on recordedliver weight and immediately homogenized using a FastPrep 24 manifold.The liver sample was then mixed with 10% by volume of1-Bromo-3-chloropropane and centrifuged for 10 minutes at 4° C. toextract total RNA. 50 μL of the total extract was subjected to RNApurification using the MagMax Microarray protocol and eluted into 100 μLelution buffer. Blood RNA was isolated using the same method except that100 μL of the extract was used for purification.

RNA samples were individually normalized to 100 ng/μl of total liver RNAand blood RNA was normalized to 50 ng/μL. 10 μL of normalized input RNAwas reverse transcribed using the High Capacity Reverse Transcriptionreagents (Life Technologies). The cDNA product was diluted 1:5 foranalysis by qPCR. A standard curve for luciferase was generated usingfree mRNA diluted to 0.05 mg/mL and then six ten-fold serial dilutions.The standard curve was reverse transcribed and diluted in the samefashion as assay samples.

Reverse Transcription cDNA product was analyzed for luciferase and GAPDHusing TaqMan gene expression assays. 8 μL of the diluted cDNA was mixedwith 10 μL of TaqMan Gene Expression Master Mix (Life Technologies) and1 μL of each primer/probe set and run with standard cycling conditions.The standard curve was used to determine absolute quantities of FLucmRNA in assay samples.

Table 26 displays luminescence values in the liver for animals treatedwith P20+FLuc mRNA, P22+FLuc mRNA, P23+FLuc mRNA, or buffer. Data wasacquired at 3 hours post dose. Luminescence values are shown as ageomean from 5 animals in each group. All 3 polymer-mRNA formulationsdemonstrated strong luminescence signal in the liver compared to buffer.Polymers P22 and P23 demonstrated 5-10 fold greater luminescence signalin the liver compared to P20.

TABLE 26 Luminescence results at 3 hours post dose Polymer mRNA DoseDose Total Flux (photons/sec) (mg/kg) (mg/kg) Geomean STDEV Buffer 0 02.13E+05 1.32E+05 P20 + FLuc 100 1 7.82E+06 4.14E+07 mRNA P22 + FLuc 501 4.67E+06 2.71E+07 mRNA 75 1 1.02E+08 2.29E+08 100 1 1.18E+08 5.27E+07P23 + FLuc 50 1 1.42E+07 1.75E+07 mRNA 75 1 5.97E+07 1.98E+08 100 13.51E+07 1.64E+08

Table 27 displays % dose FLuc mRNA in the liver and Table 28 displays %dose in blood for animals treated with P20+FLuc mRNA, P22+FLuc mRNA,P23+FLuc mRNA, buffer, or unformulated mRNA. Data was acquired at 1 and30 minutes post dose. % mRNA dose values are shown as an average from 5animals in each group. While neither buffer nor unformulated mRNA hadany detection in liver or blood, mRNA was detected in all 3 polymer-mRNAformulations. Polymers P22 and P23 demonstrated approximately 20-foldgreater FLuc mRNA delivered to the liver compared to P20 at 1 minutepost dose. P20 had significantly higher levels of FLuc mRNA in blood at1 minute compared to P22 and P23.

TABLE 27 Percent FLuc mRNA dose in liver at 1 and 30 minutes post dosePolymer mRNA Dose Dose FLuc mRNA (% dose) (mg/kg) (mg/kg) Time PointAverage STDEV Buffer 0 0 1 min 0.0% 0.0% Fluc mRNA 0 1 1 min 0.0% 0.0%P20 + FLuc 75 0.75 1 min 0.6% 0.1% mRNA 30 min 0.1% 0.0% P22 + FLuc 750.75 1 min 10.2% 8.8% mRNA 30 min 0.6% 0.3% P23 + FLuc 75 0.75 1 min14.4% 2.8% mRNA 30 min 0.4% 0.3%

TABLE 28 Percent FLuc mRNA dose in blood at 1 and 30 minutes post dosePolymer mRNA Dose Dose FLuc mRNA (% dose) (mg/kg) (mg/kg) Time PointAverage STDEV Buffer 0 0 1 min 0.0% 0.0% FLuc mRNA 0 1 1 min 0.0% 0.0%P20 + FLuc 75 0.75 1 min 24.2% 9.5% mRNA 30 min 0.2% 0.1% P22 + FLuc 750.75 1 min 3.5% 7.0% mRNA 30 min 0.1% 0.0% P23 + FLuc 75 0.75 1 min 0.3%0.1% mRNA 30 min 0.0% 0.0%

Example 27: In Vivo Testing of Polymer-mRNA Formulations

FLuc mRNA was formulated with polymers P20 and P21 as described inExample 25. Mice were injected with polymer-FLuc mRNA formulation andexamined for luciferase expression and FLuc mRNA quantification in liverand blood as described in Example 26. Table 29 displays luminescencevalues in the liver for animals treated with polymer+FLuc mRNA. Bothpolymers demonstrated strong luminescence signal in the liver comparedto buffer. Polymer P21 demonstrated ˜10 fold greater luminescence signalin the liver compared to PRX398.

TABLE 29 Luminescence results at 3 hours post dose Polymer mRNA DoseDose Total Flux (photons/sec) (mg/kg) (mg/kg) Geomean STDEV Buffer 0 02.37E+05 1.33E+05 P20 + Fluc 100 1 3.80E+06 1.03E+07 mRNA P21 + Fluc 751 2.91E+07 1.57E+08 mRNA 100 1 4.69E+07 1.26E+08

Table 30 displays % dose FLuc mRNA in the liver and Table 31 displays %dose in blood for animals treated with polymer P21+FLuc mRNA. mRNA wasdetected in liver and blood with P21 polymer-mRNA formulation. PolymerP21 showed a similar amount of FLuc mRNA delivered to the liver ascompared to polymer P20 at 1 and 30 minutes post dose shown in Example26. Polymer P21 had significantly higher levels of FLuc mRNA in blood at1 and 30 minute compared to polymer P20 in Example 26. This may indicateincreased circulatory stability in the blood with polymer P21.

TABLE 30 Percent FLuc mRNA dose in liver at 1 and 30 minutes post dosePolymer mRNA Dose Dose Fluc mRNA (% dose) (mg/kg) (mg/kg) Time PointAverage STDEV Buffer 0 0 1 min 0.0% 0.0% P21 + Fluc 75 1 1 min 0.8% 0.4%mRNA 30 min  0.1% 0.0%

TABLE 31 Percent FLuc mRNA dose in blood at 1 and 30 minutes post dosePolymer mRNA Dose Dose Fluc mRNA (% dose) (mg/kg) (mg/kg) Time PointAverage STDEV Buffer 0 0 1 min 0.0% 0.0% P21 + Fluc 75 1 1 min 79.7%22.4% mRNA 30 min  2.4% 1.4%

Example 28: Formulation of Polymers with FLuc mRNA

Polymers are solubilized in 20 mM HEPES/5% glucose, pH 7.4(HEPES/glucose) plus 20% ethanol with agitation typically between 20-40mg/mL for 1 hour at room temperature and then incubated overnight at 4°C. FLuc (firefly luciferase) mRNA stock solution at 1 mg/mL in 10 mMTris-HCL (pH7.5) from TriLink Biotechnologies is diluted to 0.2 mg/mL inHEPES/glucose buffer. Using a microfluidics device, the polymer and mRNAsolutions are mixed at a 1:1 volume, at 12 mL/minute, and at an N/Pratio typically between 10 and 20. The final concentrations of thepolymer and mRNA are typically at 2-20 mg/mL and 0.1 mg/mL respectivelyin HEPES/glucose with 10% ethanol buffer. The formulations are incubatedat room temperature for approximately 60 minutes prior to injectingmice.

The formulation particle size is measured by adding 10 μl of formulationto 90 μL of HEPES/glucose buffer into a disposable micro-cuvette andanalyzed using the Malvern Instrument ZETASIZER NANO-ZS. The formulationzeta-potential at pH 7.4 is measured by adding 10 μl of formulation to740 μL of HEPES/glucose buffer into a disposable 1 mL cuvette. The zetadip cell is inserted into the 1 mL cuvette and the formulation isanalyzed using the ZETASIZER NANO-ZS. The zeta-potential is alsomeasured at pH 4 as described above by adding 10 μl of formulation to740 μL of 20 mM acetate buffer pH 4 containing 5% glucose. The abilityof the polymer formulation to compact the mRNA is measured in a 96 wellplate using a SYBR Gold dye accessibility assay. Typically, 50 μL of thepolymer formulation at 0.01 mg/mL mRNA is added to 150 μL of dilutedSYBR Gold stock solution (1 μL of Stock SYBR Gold in 3 mL ofHEPES/glucose buffer) and incubated for 15 minutes at room temperaturewith agitation. The fluorescence is read at an excitation wavelength of495 nm and emission wavelength of 538 nm. The percent dye accessibilityis calculated by dividing the fluorescence intensity of the formulatedmRNA by the fluorescence intensity of the free mRNA×100.

Example 29: In Vivo Testing of Polymer-mRNA Formulations

FLuc mRNA was formulated with each of the following polymers: P20, P24,P31, P32, P58, P59, P62, P61, P27, P33, P34, P35, P36, P37, P38, P39,P40, P64, P65, and P66. Polymers were formulated as described in Example28, except for one study comparing polymers P58 and P59 to polymer P20,in which these polymers were formulated as described in Example 25. Micewere injected with polymer-FLuc mRNA formulations and examined forluciferase expression as described in Example 26.

Table 32 displays luminescence values in the liver for animals treatedwith polymer+FLuc mRNA in an experiment comparing polymers P24, P31, andP32 to polymer P20. Polymers demonstrated strong luminescence signal inthe liver compared to buffer. Polymer P24 demonstrated ˜7 fold greaterluminescence signal in the liver compared to polymer P20.

Table 33 displays luminescence values in the liver for animals treatedwith polymer+FLuc mRNA in an experiment comparing polymers P58 and P59to polymer P20. Polymers demonstrated strong luminescence signal in theliver compared to buffer. Polymer P58 demonstrated ˜20 fold greaterluminescence signal in the liver compared to polymer P20.

Table 34 displays luminescence values in the liver for animals treatedwith polymer+FLuc mRNA in an experiment comparing polymers P62, P61, andP27 to polymer P20. Polymers demonstrated strong luminescence signal inthe liver compared to buffer. Polymer P27 demonstrated ˜5 fold greaterluminescence signal in the liver compared to polymer P20.

Table 35 displays luminescence values in the liver for animals treatedwith polymer+FLuc mRNA in an experiment comparing polymers P33, P34,P35, and P36 to polymer P20. Polymers demonstrated strong luminescencesignal in the liver compared to buffer. Polymers P33, P34, and P35demonstrated ˜12-30 fold greater luminescence signal in the livercompared to polymer P20.

Table 36 displays luminescence values in the liver for animals treatedwith polymer+FLuc mRNA in an experiment comparing polymers P37, P38,P39, and P40 to polymer P20. Polymers demonstrated strong luminescencesignal in the liver compared to buffer. Polymer P37 demonstrated ˜4 foldgreater luminescence signal in the liver compared to polymer P20.

Table 37 displays luminescence values in the liver for animals treatedwith polymer+FLuc mRNA in an experiment comparing polymers P64, P65, andP66 to polymer P20. Polymers demonstrated strong luminescence signal inthe liver compared to buffer. Polymers P64 and P66 demonstrated ˜4 foldgreater luminescence signal in the liver compared to polymer P20.

TABLE 32 Luminescence results at 3 hours post dose Polymer mRNA DoseDose Total Flux (photons/sec) (mg/kg) (mg/kg) Geomean STDEV Buffer 0 02.08E+05 NA P20 + Fluc 100 1 2.83E+07 6.91E+07 mRNA P24 + Fluc 53 11.83E+08 1.92E+08 mRNA 80 1 1.62E+07 1.41E+08 P31 + Fluc 62 1 1.76E+076.20E+08 mRNA 93 1 1.21E+08 1.59E+08 P32 + Fluc 74 1 1.30E+06 9.23E+06mRNA 112 1 1.17E+06 4.78E+06

TABLE 33 Luminescence results at 3 hours post dose Polymer mRNA DoseDose Total Flux (photons/sec) (mg/kg) (mg/kg) Geomean STDEV Buffer 0 02.61E+05 NA P20 + Fluc 100 1 2.58E+06 7.47E+06 mRNA P58 + Fluc 100 15.09E+07 9.71E+07 mRNA P59 + Fluc 100 1 1.68E+07 8.95E+07 mRNA

TABLE 34 Luminescence results at 3 hours post dose Polymer mRNA DoseDose Total Flux (photons/sec) (mg/kg) (mg/kg) Geomean STDEV Buffer 0 01.70E+05 NA P20 + Fluc 100 1 1.53E+07 4.75E+07 mRNA P62 + Fluc 50 15.95E+05 1.40E+06 mRNA P61 + Fluc 75 1 3.51E+07 2.01E+08 mRNA P27 + Fluc108 1 7.48E+07 7.19E+07 mRNA

TABLE 35 Luminescence results at 3 hours post dose Polymer mRNA DoseDose Total Flux (photons/sec) (mg/kg) (mg/kg) Geomean STDEV Buffer 0 01.20E+05 NA P20 + Fluc 100 1 8.81E+06 9.94E+06 mRNA P33 + Fluc 38 12.74E+08 6.22E+08 mRNA 77 1 3.91E+07 2.52E+08 P34 + Fluc 68 1 1.08E+081.95E+08 mRNA 102 1 1.11E+08 1.38E+08 P35 + Fluc 74 1 1.81E+08 2.04E+08mRNA 111 1 3.85E+07 2.97E+07 P36 + Fluc 69 1 9.78E+06 1.97E+08 mRNA 1041 4.70E+07 5.43E+08

TABLE 36 Luminescence results at 3 hours post dose Polymer mRNA DoseDose Total Flux (photons/sec) (mg/kg) (mg/kg) Geomean STDEV Buffer 0 02.27E+05 NA P20 + Fluc 100 1 2.77E+07 7.04E+07 mRNA P37 + Fluc 63 11.15E+08 2.48E+08 mRNA 95 1 2.94E+08 3.87E+08 P38 + Fluc 68 1 4.31E+073.63E+08 mRNA 101 1 5.75E+07 1.72E+08 P39 + Fluc 102 1 2.16E+07 1.57E+08mRNA 153 1 1.18E+07 5.91E+07 P40 + Fluc 123 1 9.98E+05 3.90E+06 mRNA 1851 1.49E+07 7.13E+07

TABLE 37 Luminescence results at 3 hours post dose Polymer mRNA DoseDose Total Flux (photons/sec) (mg/kg) (mg/kg) Geomean STDEV Buffer 0 01.98E+05 NA P20 + Fluc 100 1 1.53E+07 4.75E+07 mRNA P64 + Fluc 77 16.01E+07 3.33E+08 mRNA 103 1 4.74E+07 3.87E+08 P65 + Fluc 76 1 2.64E+079.63E+07 mRNA 102 1 2.85E+07 1.69E+08 P66 + Fluc 66 1 5.82E+07 1.82E+08mRNA 88 1 2.00E+07 4.99E+07

Example 30: Treatment of OTCD with Polymer-mRNA Formulations inOTC-spf^(ash) Mice

OTC-spf^(ash) (sparse fur and abnormal skin and hair) mice contain anR129H mutation which results in reduced levels of OTC protein and haveonly 5-10% of the normal level of enzyme activity in liver (see Hodgeset al., PNAS 86:4142-4146, 1989). The OTC-spf^(ash) mouse model haselevated urine orotic acid levels compared to wild-type littermate mice.

Groups of 5-10 OTC-spf^(ash) mice are treated by intravenous route ofadministration with synthetic OTC mRNA formulated with polymer thattargets hepatocytes in the liver, thereby achieving expression andactivity of OTC. Mice are treated with vehicle control or OTC mRNA from0.1-5 mg/kg. Either single or repeat dosing is performed with a varietyof dosing intervals (e.g. twice daily, daily, every 2 days, etc.). Urineis collected pre-dose as well as at different time points ranging from 3hours to 48 hours post final dose on the short term or up to 2 weekspost dose for duration of expression. At these time points, mice aresacrificed and livers are collected and sampled to evaluate OTC proteinexpression by western analysis immunofluorescence of liver tissuesection, and OTC enzyme activity. Urine is analyzed for orotic acidlevels normalized to creatinine levels.

Results are compared to vehicle-treated OTC deficient mice. In addition,results are compared to wild-type litter mate mice that have normallevels of OTC protein expression, enzyme activity and urine orotic acidlevels. Efficacy is shown by detectable levels of OTC protein expressionevaluated by western and immunofluorescence that are above the leveldetected in vehicle treated mice, enzyme activity that is at least 15%of normal levels, and urine orotic acid levels are reduced at least 50%compared to vehicle control treated mice.

Example 31: Treatment of OTCD with Polymer-mRNA Formulations in OTC-spfMice

OTC-spf mice contain an H117N mutation, which results in reduced levelsof enzyme activity to 5-10% of normal levels (see Rosenberg et al.,Science 222:426-428, 1983). The OTC-spf mouse model has elevated urineorotic acid levels compared to wild-type littermate mice.

Groups of 5-10 OTC-spf mice are treated by intravenous route ofadministration with synthetic OTC mRNA formulated with polymer thattargets hepatocytes in the liver, thereby achieving expression andactivity of OTC. Mice are treated with vehicle control or OTC mRNA from0.1-5 mg/kg. Either single or repeat dosing is performed with a varietyof dosing intervals (e.g. twice daily, daily, every 2 days, etc.). Urineis collected pre-dose as well as at different time points ranging from 3hours to 48 hours post final dose on the short term or up to 2 weekspost dose for duration of expression. At these time points, mice aresacrificed and livers are collected and sampled to evaluate OTC proteinexpression by western analysis immunofluorescence of liver tissuesection, and OTC enzyme activity. Urine is analyzed for orotic acidlevels normalized to creatinine levels.

Results are compared to vehicle-treated OTC deficient mice. In addition,results are compared to wild-type litter mate mice that have normallevels of OTC protein expression, enzyme activity and urine orotic acidlevels. Efficacy is shown by detectable levels of OTC protein expressionevaluated by western and immunofluorescence that are above the leveldetected in vehicle treated mice, enzyme activity that is at least 15%of normal levels, and urine orotic acid levels are reduced at least 50%compared to vehicle control treated mice.

Example 32: Treatment of OTCD with Polymer-mRNA Formulations in aHyperammonemia Mouse Model

An additional model for OTC deficiency is inducing hyperammonemia inOTC-spf or OTC-spf^(ash) mice (see Cunningham et al., Mol Ther 19(5):854-859, 2011). These mice are treated with OTC siRNA or AAV2/8vector/OTC shRNA to knockdown residual endogenous OTC expression andactivity. Plasma ammonia levels are elevated and mice die approximately2-14 days.

Groups of 5-10 hyperammonemia-induced mice are treated by intravenousroute of administration with synthetic OTC mRNA formulated with polymerthat targets hepatocytes in the liver, thereby achieving expression andactivity of OTC. Mice are treated with vehicle control or OTC mRNA from0.1-5 mg/kg. Either single or repeat dosing is performed with a varietyof dosing intervals (e.g. twice daily, daily, every 2 days, etc.).Animals are monitored for ataxia, a clinical sign of hyperammonemia,and >10% body weight loss over 24 hours starting at 24 hours and up to 4weeks post dose. Blood and urine are collected from mice that developataxia to examine plasma ammonia levels and orotic acid levels.Immediately following, mice are sacrificed and livers are collected andsampled to evaluate OTC protein expression by western analysis,immunofluorescence of liver tissue sections, and OTC enzyme activity.

Results are compared to vehicle-treated OTC deficient mice. In addition,results are compared to wild-type litter mate mice that have normallevels of OTC protein expression, enzyme activity and urine orotic acidlevels. Efficacy is shown by increased survival compared to vehiclecontrol treated mice and at least 50% reduction in plasma ammonialevels. Efficacy is also indicated by detectable levels of OTC proteinexpression evaluated by western and immunofluorescence that is above thelevel detected in vehicle treated mice, enzyme activity that is at least15% of normal levels, and urine orotic acid levels are reduced at least50% compared to vehicle control treated mice.

Example 33: Treatment of MMA with Polymer-mRNA Formulations in Mut^(−/−)Mice or Mut^(−/−); Tg^(INS-MCK-Mut) Mice

Groups of 5-10 Mut^(−/−); Tg^(INS-MCK-Mut) mice are treated byintravenous route of administration with synthetic mRNA formulatedpolymer that targets hepatocytes in the liver, thereby achievingexpression and activity of MUT protein. Mice are treated with vehiclecontrol or Mut mRNA from 0.1-5 mg/kg. Either single or repeat dosing isperformed with a variety of dosing intervals (e.g. twice daily, daily,every 2 days, etc.). Plasma is collected to examine methylmalonic acidlevels at different time points ranging from 3 hours to 72 hours postfinal dose on the short term or up to 2 weeks post dose for duration ofexpression. A ¹³C propionate oxidation/breathe assay is performed atdifferent time points ranging from 24-72 hours post dose or longer timepoints up to 2 weeks post dose to examine in vivo metabolic effects ofMUT protein expression. At these time points, mice are sacrificed andlivers are collected and sampled to evaluate MUT protein expression bywestern analysis and immunofluorescence of liver tissue sections.

Results are compared to vehicle-treated mice as well as to wild-typelitter mate mice that have normal levels of MUT protein expression,methylmalonic acid levels in plasma, and ¹³C propionate oxidation.Efficacy is shown by detectable levels of MUT protein expressionevaluated by western and immunofluorescence that is above the leveldetected in vehicle treated mice. Plasma methylmalonic acid levels arenormally <5 μM in wild-type littermate mice whereas Mut^(−/−);Tg^(INS-MCK-Mut) mice have 200-400 μM methylmalonic acid levels.Efficacy by plasma methylmalonic acid levels is a correction towardslevels seen in wild-type littermate mice. Efficacy is also detected by asignificant increase in ¹³C propionate oxidation compared to vehiclecontrol treated mice.

Example 34: Treatment of PA with Polymer-mRNA Formulations in Pcca^(−/−)(A138T) Mice

Groups of 5-10 Pcca^(−/−) (A138T) mice are treated by intravenous routeof administration with synthetic mRNA formulated polymer that targetshepatocytes in the liver, thereby achieving expression and activity ofPCC enzyme. Mice are treated with vehicle control or Pcca mRNA from0.1-5 mg/kg. Either single or repeat dosing is performed with a varietyof dosing intervals (e.g. twice daily, daily, every 2 days, etc.). Bloodis collected to examine propionylcarnitine/acetylcarnitine ratio,methylcitrate, and plasma ammonia levels at different time pointsranging from 3 hours to 72 hours post final dose on the short term or upto 2 weeks post dose for duration of expression. At these time points,mice are sacrificed and livers are collected and sampled to measure PCCenzyme activity and protein expression.

Results are compared to vehicle-treated mice as well as to wild-typelitter mate mice that have normal PCC enzyme activity, metabolite levelsin blood, and protein expression. Efficacy is shown by detectable levelsof PCCA protein evaluated by western that is above the level detected invehicle treated mice. PCC enzyme activity is normally ˜2% of wild-typelevels in affected mice. PCC enzyme activity at 10-20% of wild-typelevels or higher is seen as efficacious. Reduction inpropionylcarnitine/acetylcarnitine ratio, methylcitrate, and plasmaammonia levels also demonstrate efficacy.

Example 35: Reaction of NAG-C5-PEG₃₆NH2 with Polymer P19:NAG-PEG-₁₂-[PEGMA (300, 73%)-TFPMA (27%)]4.55 KDa-b-[DMAEMA (36%)-BMA(46%)-PAA (10%)-PDSMA (7%)]5.33 KDa

To polymer P19 (175 mgs, 0.000016 mole) in a 40m1 glass vial was addedDMF (1 mL) at RT under argon. To the solution was added NAG-C5-PEG₃₆NH2(250 mgs, 0.000128 mole) and the mixture was stirred until a solutionwas obtained (15 min). TEA (19 ul, 0.000156 mole) was added and theyellow solution was heated to 60 C and held at that temperature for 48hrs. The reaction was then treated with 3-amino-1-propanol (17 uL) andheating at 60 C was continued for 7 hrs. The reaction was diluted withAcetone (2 mL) and the product was precipitated with Et2O/Hexanes (1:3,40 mL). The precipitation was repeated twice more. The resultant pelletwas dried under high vacuum for 15 hrs.

The resulting pellet was dissolved in MeOH (3 mL) and the solution wastreated with 0.5M TCEP (500 ul, 10 equiv). After 30 min the yellowsolution was treated with DPDS (112 mgs, 20 equiv). The reaction wasagitated for 60 min then the solution was diluted with MeOH (12 ml) andwas dialyzed from MeOH thrice. The contents of the dialysis bag wereconcentrated on the rotavap and the residue dried for 16 hrs. HNMRshowed ˜4 out of 5.5 TFP units were displaced by NAGPEG₃₆NH2, to affordpolymer P30: NAG-PEG₁₂-[PEGMA (300, 73%)-NAG-C5-PEG₃₆ (18%)-TFPMA(5%)]11 KDa-b-[DMAEMA (36%)-BMA (46%)-PAA (10%)-PDSMA (7%)]5.33 KDa.

By similar process, polymer P18: ECT-[PEGMA (300, 58)-TFPMA (42)] 5.14KDa-b-[DMAEMA (31)-BMA (49)-PAA (12)-PDSMA (8)] 5.03 KDa, was modifiedwith NAG-C5-PEG₃₆NH2, to afford polymer P31 ECT-[PEGMA (300,58)-NAG-C5-PEG₃₆ (42)] 19.9 KDa-b-[DMAEMA (31)-BMA (49)-PAA (12)-PDSMA(8)] 5.03 KDa.

Example 36: Synthesis of Tri-NAG CTA Part 1: Synthesis ofFmoc-Amido-Triacid

Procedure:

To a 100 mL round bottom flask was added FmocCl (3.14 g, 12.2 mmol).This material was dissolved in THF (50 mL). In a separate 250 mL roundbottom flask was addeddi-tert-butyl-4-amino-4-[2-(tertbutoxycarbonyl)ethyl]heptanedionate(5.00 g, 12.0 mmol, tris t-butyl amine) which was then dissolved in THF(50 mL). The FmocCl solution was then added to the solution of trist-butyl amine in one portion. Immediately following the addition,triethylamine (1.79 mL, 12.8 mmol) was added to the reaction mixture.Upon the addition of triethylamine a precipitate formed in the flask.The reaction was left to stir under argon atmosphere at room temperaturefor 30 minutes.

The crude reaction mixture was concentrated using rotary evaporation.The product was used as a crude mixture for the next reaction in thesynthetic sequence (assuming quantitative yield).

To the crude reaction mixture was added TFA (25 mL). A white solidremained in the reaction mixture. This mixture was concentrated byrotary evaporation until approximately 5 mL remained. To the mixture wasadded water (1 mL) to react any trifluoroacetic anhydride and convert itto carboxylic acid. The mixture was concentrated by rotary evaporationand high vacuum, resulting in white solid. This solid was trituratedwith ethyl acetate and the solid was centrifuged. The solid wastriturated and centrifuged until 6.4 g (84%, two steps) colorless solidwith good purity was recovered.

Part 2: Synthesis of Fmoc-Amido-tri-pentafluorophenyl ester

Procedure:

To a 250 mL one-neck round bottom flask was added TL-02-19 (3.3 g, 7.0mmol, thoroughly dried overnight on high vacuum) followed by anhydrousTHF (60 mL, lot # B0313244). This mixture was stirred under a flow ofargon gas and then cooled to 0° C. for 5 min. Then trifluoroacetic acidpentafluorophenyl ester (4.0 mL, 23.2 mmol, lot #69096MJ) was added dropwise followed by triethylamine (3.24 mL, 23.2 mmol, lot # B0518226). Thereaction was then warmed to room temperature under a flow of argon gas.

The reaction progress can be followed by TLC (SiO₂, 100% CH₂Cl₂) bylooking for the disappearance of the starting material TL-02-19 (Rf=0.0)and the appearance of the PFP activated product MD-03-20 (Rf=0.49).After stirring for 2.0 h at room temperature the starting material wascompletely consumed by TLC.

Once the starting material was consumed by TLC the crude reaction wasevaporated using a rotary evaporator to remove all the THF. Once thecrude reaction was condensed to a viscous oil the mixture was dissolvedwith CH₂Cl₂ (150 mL) and extracted using saturated aqueous NaHCO₃ (3×50mL). The organic layer was separated, dried over Na₂SO₄, filtered andevaporated providing 6.0 g (89%) of the final product as a white solid.All solvents and volatile reagents were thoroughly removed using highvacuum (0.5 mmHg) overnight before the crude product was used in thenext synthetic step. No characterization of the final product waspreformed other than TLC analysis (TLC conditions described above). TheTLC analysis of the final product showed the material was only onecompound (Rf=0.49). Ninhydrin TLC based (300 mg ninhydrin dissolved in100 mL EtOH and 3 mL AcOH) analysis showed that there was no Fmocdeprotected product produced via this process.

Part 3: Synthesis of Fmoc-Amido Triacid-2

Procedure:

To a 250 mL one-neck round-bottom flask was added MD-03-20 (6.0 g, 6.2mmol) followed by anhydrous DMSO (50 mL). This mixture was stirred atroom temperature under a flow of inert argon gas until MD-03-20 wascompletely dissolved. Then (2-amino-ethoxy)-acetic acid (2.26 g, 18.97mmol, compound acquired from Chess Fine Organics) was added directly tothe reaction mixture. The (2-amino-ethoxy)-acetic acid slowly dissolvedinto the reaction mixture. After all reagents were dissolved the mixturewas stirred at room temperature under a flow of argon gas for 2 h.

The reaction progress can be followed by analytical HPLC by diluting thereaction mixture (10 μL) into DMSO (700 μL) and injecting 10 μL of thatdiluted mixture (FIG. 1). The HPLC analysis was determined usingShimadzu LD-20AB with the UV detector set to 210 nm through a C18analytical reverse phase column (ES Industries Chromega Columns, SonomaC18 catalog number 155B21-SMA-C18(2), 100 Å, 25.0 cm×4.6 mm, columnheated to 30.0° C., CH₃CN/H₂O containing 0.01% TFA, isocratic gradientat 10% CH₃CN for 2 min, then linear gradient from 10% to 60% CH₃CN over20 min, total flow rate of 1.0 mL/min). The desired tri acid product hasa retention time of 16.45 min, the pentafluorophenol (PFP) leaving groupwas found at 22.1 min, and the loss of Fmoc was detected at 24.7 min.

After reacting 2 h at room temperature the reaction was complete. Thecrude reaction was directly purified using C18 preparative reverse phaseHPLC by Shimadzu (Phenomenex, Luna 5 C18(2), part number 00G-4252-P0-AX,100 Å, 25.0 cm×21.2 mm, with a SecurityGuard PREP Cartridge, C18 15×21.2mm ID, part number AJ0-7839, CH₃CN/H₂O with 0.01% TFA, isocraticgradient at 10% CH₃CN for 5 min, then linear gradient from 10% to 35%CH₃CN over 15 min, then linear gradient from 35% to 40% CH₃CN over 5min, then isocratic gradient at 40% CH₃CN for 2 min, total flow rate of20.0 mL/min, column at room temperature). Roughly 1.5 mL of the crudereaction mixture in DMSO (˜100 mg/mL) were injected each HPLC run. Usingthe HPLC purification conditions above the desired product elutedbetween 24.5 and 25.5 min. The fraction(s) associated with the desiredproduct were pooled together, and the solvent thoroughly evaporatedusing a rotary evaporator. Then the final product was transferred to aflask using MeOH and all solvents were completely removed using highvacuum (pressure <0.5 mmHg) overnight providing 3.13 g (65%) of thedesired product as a white solid.

The final product was dissolved in MeOH (ca. 1. mg/mL) and analyzed byC18 analytical reverse phase HPLC. The HPLC analysis was determinedusing Shimadzu LD-20AB with the UV detector set to 210 nm through a C18analytical reverse phase column (ES Industries Chromega Columns, SonomaC18 catalog number 155B21-SMA-C18(2), 100 Å, 25.0 cm×4.6 mm, columnheated to 30.0° C., CH₃CN/H₂O containing 0.01% TFA, isocratic gradientat 10% CH₃CN for 2 min, then linear gradient from 10% to 60% CH₃CN over20 min, total flow rate of 1.0 mL/min). The final product has aretention time of 16.45 min.

The final product was also analyzed using a 300 MHz ¹H NMR with CD₃OD assolvent and is consistent with the structure. The final product was alsoanalyzed using Bruker Esquire Ion Trap Mass Spectrometer showing theM+Na ion (m/z=795.8) and the 2M+Na ion (m/z=1568.6).

Part 4: Synthesis of NAG(OAc)4-C5 Amine

1. Reaction 1: Synthesis of Ac-Galactosamine-C₅-NHBoc (MQ-02-70)

Raw Materials

Step 1

TABLE 38 MW (density Vol g/mL) Weight Moles equiv (mL) Lot# Supplier2-acetamido- 389.35 19.2 g 0.049 1 MA07898110 Carbosynth1,3,4,6-tetra-O- acetyl-2-deoxy- D- galactopyranose DCM, 300 BOON0138Acros anhydrous 34846-1000 Trimethylsilyltrifuoromethane- 222.26 0.1182.4 21.4 BCBB3628V Fluka sulfonate (1.23) 91741-50 mL (TMSOTf)Triethylamine 101.2 0.0686 1.4 9.64 SHBC1859V Aldrich (TEA) (0.726)T0886-100 mL

Step 2

TABLE 39 MW (density Vol g/mL) Weight Moles equiv (mL) Lot# SupplierOxazoline 329.20 17.57 0.049 1 intermediate 5- 203.4 14.75 g 0.0725 1.48SQCQJ-TM TCI B2869 (tbutoxycarbonylamino)- >97% 1- pentanol DCM, 300BOON0138 Acros anhydrous 34846-1000 Trimethylsilyltrifuoromethane-222.26 0.024 0.49 4.34 BCBB3628V Fluka sulfonate (1.23) 91741-50 mL(TMSOTf) Triethylamine 101.2 0.034 0.7 4.82 SHBC1859V Aldrich (TEA)(0.726) T0886-100 mL

Synthesis

Step 1

Anhydrous DCM (300 mL) was added to2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-galactopyranose (19.2 g,0.049 mole) in an oven-dried 500 mL round-bottom flask (RBF) at RT underargon. TMSOTf (21.4 mL, 0.118 mole, 2.4 equiv) was then added in oneportion to the thin suspension at RT. The RBF was fitted with a refluxcondenser and was heated in an oil bath to bring the reaction to reflux.After 4.5 hr, thin layer chromatography (TLC) (80% EtOAc/Hexanes,visualization using KMnO₄ dip, product is less polar than startingmaterial, product Rf=0.40; SM Rf=0.33) showed reaction completion.Heating was stopped and the reaction flask was allowed to reach RT. Thereaction flask was placed in an ice bath for 15 min and the reaction wasquenched with triethylamine (9.64 mL, 0.069 mole, 1.4 equiv), and theresulting solution was stirred for 15 min. The DCM layer was then washedwith sat NaHCO₃ (2×125 mL), brine (1×150 mL), H₂O (1×150 mL) and driedover Na₂SO₄ (34 g) with stirring for 1 hr. The golden yellow solutionwas filtered and concentrated under reduced atmosphere (bath temp 29°C.). This material can be stored at −20° C. without any deleteriouseffects. The residue was placed under high vacuum for 2 hr to give athick syrup (17.57 g) which was used as is in the next step.

Step 2

Anhydrous DCM (200 mL) was added to the oxazoline intermediate (obtainedin step 1) at RT under argon. When complete dissolution was achieved asolution of 5-(t-butoxycarbonylamino)-1-pentanol) dissolved in anhydrousDCM (100 mL) was added. 4 Å molecular sieves (powdered, used as receivedfrom supplier) were added at RT and the suspension was stirred for 1 hrunder argon. TMSOTf (4.34 mL, 0.024 mole, 0.49 equiv) was added in oneportion at RT and the suspension was stirred at RT overnight. After 21hr at RT the reaction flask was placed in an ice bath for 25 min, thentriethylamine (4.82 mL, 0.034 mole, 0.7 equiv) was added over 1 min.After 30 min the ice bath was removed and the reaction was allowed toreach RT over 30 min. The reaction suspension was filtered through a padof celite and the celite was washed with additional DCM (75 mL). Thecombined DCM layer was washed with H₂O (1×150 mL), sat NaHCO₃ (1×150mL), H₂O (1×150 mL) and dried over Na₂SO₄ (23 g) for 1 hr. The solutionwas filtered and DCM was removed under reduced atmosphere. The residuewas dried under high vacuum for 7 hr.

Flash Chromatography

Column Specifications

-   -   Dry silica gel: 1250 mL    -   Slurrying solvent: 50% EtOAc/Hexanes    -   Column dimensions: 11.5 cm×14 cm (Diameter×Height)    -   Flow rate: ˜3 L/hr    -   Eluent:

50% EtOAc/Hexanes 3 L 80% EtOAc/Hexanes 5 L 100% EtOAc/Hexanes 2 L

Elution and Fraction Collection

After 5.5 L had eluted, fraction collection was started. A total of17×250 mL fractions were collected. The pure product was in fractions 2to 15. The pure fraction was concentrated on a rotavap to give a whitewaxy solid. The solid was dried under high vacuum for 24 hr to give18.71 g (72% yield for 2 steps) of Ac-Galactosamine-C₅-NHBoc (MQ-02-70).

¹H-NMR (CD₃OD, 400 MHz) of Ac-Galactoseamin-C₅-NHBoc (MQ-02-70):Results: NMR looks consistent for proposed structure.

Part 5: Synthesis of Fmoc-amido-tri-NAG(OAc)₄

Procedure:

Step 1. Sugar Deprotection:

To a 250 mL one-neck round-bottom flask was added TL-01-74 (9.08 g, 17.1mmol) followed by trifluoroacetic acid (50 mL, TFA). Once all of thecarbohydrate was completely dissolved the TFA was evaporated using arotary evaporator until a light yellow oil is produced. The carbohydratewas triturated with diethyl ether (Et₂O, 4×75 mL). Following the finaltrituration, the remaining was removed using a rotary evaporator thenthe crude carbohydrate is placed onto a high vacuum line (pressure ˜0.5.mmHg) overnight. After high vacuum overnight the process provided 8.75 g(94%) of the deprotected sugar.

Step 2. Coupling Reaction

Coupling Reaction:

To a 100 mL one-neck round bottom flask was added the purified triacidMD-03-21 (1.33 g, 1.72 mmol). Then the sugar TL-01-74 deprotected (8.47g, 15.5 mmol) dissolved in anhydrous DMSO (15 mL) was added to thereaction vial containing the triacid (MD-03-21). Any residual sugar waswashed with additional DMSO (5 mL) and transferred into the reactionmixture. Then 4-(dimethylamino)pyridine (11.0 mg, 0.09 mmol, DMAP) wasadded to the reaction followed by N-hydroxysuccinimide (1.78 g, 15.5mmol, NHS). This mixture was stirred vigorously at room temperaturewhile N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (2.75 mL, 15.5mmol, EDC) was added dropwise to the reaction over ca. 5 min.

The reaction progress can be followed by analytical HPLC by diluting thereaction mixture (10 μL) into a solution of MeOH (500 μL) with H₂O (200μL L) and injecting 50 μL of that diluted mixture. The HPLC analysis wasdetermined using Shimadzu LD-20AB with the UV detector set to 210 nmthrough a C18 analytical reverse phase column (ES Industries ChromegaColumns, Sonoma C18 catalog number 155B21-SMA-C18(2), 100 Å, 25.0 cm×4.6mm, column heated to 30.0° C., CH₃CN/H₂O containing 0.01% TFA, isocraticgradient at 10% CH₃CN for 2 min, then linear gradient from 10% to 60%CH₃CN over 20 min, total flow rate of 1.0 mL/min). The triacid startingmaterial MD-03-21 has a retention time of 16.4 min and the desiredproduct MD-03-24 has a retention time of 19.8 min using the above HPLCanalysis. The intermediate where only one sugar has been added has aretention time of 17.9 min while the intermediate where two sugars areadded has a retention time of 19.0 min. This reaction used the freeamine form of EDC which can cause the removal of Fmoc protecting group.In this particular process only a small amount of the product where Fmocwas deprotected was detected using this HPLC analysis. This Fmocfragment had a retention time of 26.6 min. Furthermore, the otherfragment from Fmoc removal, the tri-sugar without Fmoc, has a retentiontime of 13.5 min using the above HPLC conditions.

Analysis after 3-4 hrs indicated the reaction was not complete, andadditional N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (900 μL, 5.1mmol, EDC) was added. After the additional 900 μL of EDC was added thereaction was still incomplete by HPLC analysis. Subsequently,incremental additions of EDC were made throughout the day until most ofthe starting material and intermediates were consumed by HPLC. Thereaction was quenched after a total of 1800 μL (10.2 mmol) of additionalEDC had been added to the reaction mixture (therefore, the total amountof EDC needed to drive this reaction to completion was 4.55 mL (25.7mmol)). The reaction was quenched by the addition of H₂O (30 mL) andMeOH (20 mL), and the quenched reaction was then frozen at −80° C. untilHPLC purification.

The quenched reaction was thawed and the crude product was purifiedusing C18 preparative reverse phase HPLC by Shimadzu (Phenomenex, Luna 5C18(2), part number 00G-4252-P0-AX, 100 Å, 25.0 cm×21.2 mm, with aSecurityGuard PREP Cartridge, C18 15×21.2 mm ID, part number AJ0-7839,CH₃CN/H₂O without any additive, isocratic gradient at 30% CH₃CN for 5min, then linear gradient from 30% to 53% CH₃CN over 20 min, total flowrate of 20.0 mL/min, column at room temperature). Roughly 1.0 mL of thecrude compound dissolved in H₂O, DMSO and MeOH (3:2:2 v/v; ca. 50 mg/mL)were injected each HPLC run. Using the HPLC purification conditionsabove the desired product MD-03-24 eluted between 20.1 and 21.2 min. Thefraction(s) associated with the desired product were pooled together.All the fractions containing the desired product were combined and thesolvent thoroughly evaporated using a rotary evaporator. Then theproduct was transferred to a vial and all solvents were completelyremoved using high vacuum for >2 hours providing 2.34 g (67%) of thedesired product as a white solid.

The final product was dissolved in MeOH (ca. 1. mg/mL) and analyzed byC18 analytical reverse phase HPLC using the HPLC conditions describedabove.

The final product was also analyzed using a 300 MHz ¹H NMR with CD₃OD assolvent, and is consistent for the structure. The glycosidic linkage was100% pure beta as determined by the NMR analysis of the anomerichydrogen signal (4.54 ppm, doublet, J=8.4 Hz, 3 hydrogen) since thissignal has a large coupling constant (i.e., 8.4 Hz). The anomerichydrogen signal at 4.54 ppm integrates to 3 protons and the aromaticsignals from the Fmoc protecting group between 7.30 and 7.85 ppmintegrate to 8 protons providing high confidence that there are exactly3 N-acetylgalactosamine molecules in this structure.

The final product was also analyzed using Bruker Esquire Ion Trap MassSpectrometer, indicating a M+1=2039, and an M+K=2054.9

Part 6: Synthesis of Tri-NAG(OAc)₄-PEG₁₂-ECT

Procedure:

To a 100 mL one-neck round-bottom flask was added MD-03-30 (700 mg, 0.68mmol, extracted product) followed by anhydrous acetonitrile (2.5 mL, Lot#: B00J7229) and triethylamine (285 μL, 2.04 mmol, Lot #: A0270061). Themixture was stirred under a flow of argon gas until all of MD-03-30 wasdissolved. Then the flask was cooled to 0° C. with an ice bath. To aseparate flask was added MD-03-29 (1.13 g, 0.62 mmol) followed byanhydrous acetonitrile (2.5 mL, Lot #: B00J7229). This solution wasstirred under a flow of argon until MD-03-29 was completely dissolved.Then the solution containing MD-03-29 was added to the reaction mixtureat 0° C. drop wise over 5 min. The reaction was allowed to warm to roomtemperature and then it was stirred at room temperature overnight.

After reacting for 2 days the crude reaction was thoroughly evaporatedusing a rotary evaporator then placed under high vacuum >1 hour. Afterthe reaction was completely evaporated the crude product was dissolvedin DMSO (ca. 15 mL) and purified using C18 preparative reverse phaseHPLC by Shimadzu (Phenomenex, Luna 5 C18(2), part number 00G-4252-P0-AX,100 Å, 25.0 cm×21.2 mm, with a SecurityGuard PREP Cartridge, C18 15×21.2mm ID, part number AJ0-7839, CH₃CN/H₂O without any additive, isocraticgradient at 30% CH₃CN for 5 min, then linear gradient from 30% to 53%CH₃CN over 20 min, total flow rate of 20.0 mL/min, column at roomtemperature). Roughly 1.0 mL of the crude compound dissolved in DMSO(ca. 100 mg/mL) were injected each HPLC run. Using the HPLC purificationconditions above the desired product MD-03-31 eluted between 21.6 and23.0 min. The fraction(s) associated with the desired product werepooled together, and the water/CH₃CN solvent was completely removedafter each HPLC run using a rotary evaporator. The temperature of thewater bath on the rotary evaporated was not allowed to reach atemperature higher than 30° C. in order to help preservetrithiocarbonate group on the CTA. After the crude reaction wascompletely purified and all fractions were combined with the solventsremoved by rotary evaporation the condensed orange oil final product wasdissolved in MeOH and transferred equally to three glass vials. Theglass vials where placed under high vacuum (pressure <0.5 mmHg)overnight. The combined yield of the final product after overnightvacuum was 612 mg (37%). The bright orange oily solid product wascovered with inert argon gas, the lid was screwed on tight, the vialswere further sealed with parafilm and stored in the −80° C. refrigeratoruntil used by the polymer synthesis group.

The final product was dissolved in MeOH (ca. 1. mg/mL) and analyzed byC18 analytical reverse phase HPLC—the desired product had a retentiontime of 20.77 min. 1H-NMR (400 MHz, CD₃OD) was consistent for product.The final product was also analyzed using Bruker Esquire Ion Trap MassSpectrometer: M+2Na=1342.2 m/z, M+2H+Na=885.7 m/z.

Thus, embodiments of the block copolymers are disclosed. Theimplementations described above and other implementations are within thescope of the following claims. One skilled in the art will appreciatethat the present disclosure can be practiced with embodiments other thanthose disclosed. The disclosed embodiments are presented for purposes ofillustration and not limitation, and the present invention is limitedonly by the claims that follow.

We claim:
 1. A synthetic oligonucleotide comprising: a nucleic acidsequence encoding a functional human ornithine transcarbamylase (OTC),wherein the nucleic acid sequence comprises a codon optimized mRNAsequence at least 95% identical to nucleic acids 48-1112 of SEQ ID NO:114.
 2. The synthetic oligonucleotide of claim 1, wherein the functionalhuman OTC comprises an amino acid sequence having at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100%sequence identity with amino acid residues 35-354 of SEQ ID NO:107. 3.The synthetic oligonucleotide of claim 2, wherein the functional humanOTC further comprises a mitochondrial leader sequence.
 4. The syntheticoligonucleotide of claim 1, wherein the functional human OTC comprisesthe amino acid sequence of SEQ ID NO:107.
 5. The syntheticoligonucleotide of claim 1, wherein the codon optimized mRNA sequencecomprises nucleic acids 48-1112 of SEQ ID NO:
 114. 6. The syntheticoligonucleotide of claim 1 which comprises a sequence at least 95%identical to SEQ ID NO:
 114. 7. The synthetic oligonucleotide of claim 1which comprises a sequence at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5% or 100% identical to SEQ ID NO:
 114. 8. Thesynthetic oligonucleotide of claim 1 which comprises the sequence of SEQID NO:
 114. 9. A composition comprising: (a) the syntheticoligonucleotide of claim 1; and (b) a pharmaceutically acceptablediluent or carrier.
 10. The composition of claim 9, further comprising apeptide, polymer, and/or a lipid.
 11. The composition of claim 10,wherein the polymer comprises a block copolymer.
 12. The composition ofclaim 9, wherein the functional human OTC comprises the amino acidsequence of SEQ ID NO:107.
 13. The composition of claim 9, wherein thecodon optimized mRNA sequence comprises nucleic acids 48-1112 of SEQ IDNO:
 114. 14. The composition of claim 9, wherein the syntheticoligonucleotide comprises a sequence at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5% or 100% identicalto SEQ ID NO:
 114. 15. An oligonucleotide construct comprising: (a) anucleic acid sequence encoding a functional human ornithinetranscarbamylase (OTC), wherein the nucleic acid sequence comprises acodon optimized mRNA sequence at least about 95% identical to nucleicacids 48-1112 of SEQ ID NO: 114; and (b) a 3′ polyA tail.
 16. Theoligonucleotide construct of claim 15, wherein the functional human OTCcomprises the amino acid sequence of SEQ ID NO:107.
 17. Theoligonucleotide construct of claim 15, wherein the codon optimized mRNAsequence comprises nucleic acids 48-1112 of SEQ ID NO:
 114. 18. Theoligonucleotide construct of claim 15 which comprises a sequence atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5% or 100% identical to SEQ ID NO:
 114. 19. A method forincreasing the amount of ornithine transcarbamylase (OTC) in a livercell comprising transfecting to the liver cell a composition comprisingan oligonucleotide construct comprising a nucleic acid sequence encodinga functional human OTC and a mitochondrial leader sequence, wherein thenucleic acid sequence comprises a codon optimized mRNA sequence at leastabout 95% identical to nucleic acids 48-1112 of SEQ ID NO: 114, whereinwhen the liver cell is in a subject the composition is administered tothe subject intravenously, such that the amount of OTC in the liver cellis increased.
 20. The method of claim 19, wherein the functional humanOTC comprises the amino acid sequence of SEQ ID NO:107.
 21. The methodof claim 19, wherein the codon optimized mRNA sequence comprises nucleicacids 48-1112 of SEQ ID NO:
 114. 22. The method of claim 19, wherein theoligonucleotide construct comprises a sequence at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100%identical to SEQ ID NO:
 114. 23. The method of claim 19, wherein thecomposition further comprises a peptide, polymer, and/or a lipid. 24.The method of claim 23, wherein the polymer comprises a block copolymer.25. A method for treating ornithine transcarbamylase deficiency (OTCD)or treating or reducing the risk of hyperammonemia in a subject withOTCD comprising intravenously administering to a subject in need thereofa therapeutically effective amount of a composition comprising anoligonucleotide construct comprising a nucleic acid sequence encoding afunctional human ornithine transcarbamylase (OTC) and a mitochondrialleader sequence, wherein the nucleic acid sequence comprises a codonoptimized mRNA sequence at least about 95% identical to nucleic acids48-1112 of SEQ ID NO: 114, wherein an effective amount of the nucleicacid is transfected into the liver, such that the OTCD is treated, thehyperammonemia is treated, and/or the risk of hyperammonemia is reducedin the subject.
 26. The method of claim 25, wherein the codon optimizedmRNA sequence comprises nucleic acids 48-1112 of SEQ ID NO:
 114. 27. Themethod of claim 25, wherein the oligonucleotide construct comprises asequence at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5% or 100% identical to SEQ ID NO:
 114. 28. Themethod of claim 25, wherein the composition further comprises a peptide,polymer, and/or a lipid.
 29. The method of claim 25, wherein the subjectis a human.
 30. The method of claim 25, wherein the functional human OTCcomprises the amino acid sequence of SEQ ID NO: 107.